Test fluid collection system

ABSTRACT

The present invention relates generally to methods and materials pertaining to assays, for example immunoassays, for biomarkers in body fluids e.g. blood. The invention also relates to diagnostic or screening methods for infections, and methods of differentiating between infectious and non-infectious conditions in mammals, particularly equines, for monitoring response to anti-infective/antibiotic therapy. The invention further relates to a test fluid collection system adapted to permit dilution and analysis of the collected test fluid. The invention further relates to monitoring exertional rhabdomyolysis in equines, and assay devices for all these things.

This application is the U.S. National Stage of International ApplicationNo. PCT/IB2014/058785, filed 4 Feb. 2014, which designates the U.S.,published in English, and claims priority under 35 U.S.C. §§ 119 or365(c) to GB Application No. 1314232.8 filed 8 Aug. 2013 and GBApplication No. 1301951.8 filed 4 Feb. 2013. The entire teachings of theabove applications are incorporated herein by reference.

TECHNICAL FIELD

The present invention relates generally to methods and materialspertaining to assays, for example immunoassays, for biomarkers in bodyfluids. The invention also relates to diagnostic or screening methodsfor infections, and methods of differentiating between infectious andnon-infectious conditions in mammals, such as equines, for monitoringresponse to anti-infective/antibiotic therapy. The invention furtherrelates to a body fluid collection system adapted to permit dilution andanalysis of the collected body fluid. The invention further relates tomonitoring exertional rhabdomyolysis in equines, and assay devices forall these things.

BACKGROUND ART

Within the animal kingdom, horses are considered to be elite athletesdue to their unique physiology. Since sport horses are required toperform at an extremely high level, even small changes in health can bedetrimental to their performance and may have the potential to negatemonths of a costly training program. Further training of horses in whicha decline in performance is noted may lead to rapid degeneration incertain circumstances and therefore an indication of the cause of illhealth should be rapidly investigated. The detection of such changes isoften challenging and requires the utilization of equipment and skillwhich are typically only provided in a laboratory setting.

Common causes of poor performance that may be managed at the horse'sside by the veterinarian and the trainer include effects of themusculoskeletal system, the respiratory system, inflammation (such asgeneral inflammation or infectious inflammation) and other illness.Conditions affecting the musculoskeletal and respiratory systems are ofparticular interest as breakdown in the physiology of these systems hasbeen identified as the main cause of disruption and interruption ofthoroughbred racing competitions (Wisher et al. 1996).

In many cases, the lack of clearly visible symptoms means that thespecific body system affected cannot be determined, while in others thepoint at which the onset of symptoms becomes notable is already beyondthe threshold where an intervention can be used to preserve theperformance of the horse.

There is therefore a need for the rapid pre- or post-symptomaticdetection and early stage diagnosis of clinical and sub-clinicalinfectious conditions such as those resulting in inflammation, and todistinguish such conditions from other inflammatory conditions so thatappropriate treatment can be given. Early stage diagnosis in suchsituations may be enhanced by maintaining a careful and precise historysupported by scientific evaluation of the affected body systems.

In addition, when a problem is known or suspected with the horse, thereis a need for regular monitoring of the severity of the problem, and theresponse to intervention(s), such as response to antibiotic oranti-infective treatment, or monitoring severity of exertionalrhabdomyolysis to avoid musculoskeletal damage and over-exertion.

Further, there is a need for pre-performance screening for competitionsand for official veterinary inspections. Furthermore there would be abenefit in screening newborns for infection.

There are a number of devices which are available for point of caretesting however these devices are tools solely for the use of theveterinarian and are not designed to facilitate use by other equineprofessionals such as the trainer. Nor are they designed for diagnosisof equine disease or ER specifically. Examples of such instrumentsinclude those disclosed in U.S. Pat. No. 5,096,669 and U.S. Pat. No.5,122,284. U.S. Pat. No. 5,096,669, incorporated herein by reference,discloses a system comprising a disposable device and handheld readerwhich can perform a variety of electrochemical measurements on blood orother fluids. The system has found use in the human clinical setting andhas subsequently been adapted for veterinary use. U.S. Pat. No.5,122,284, incorporated herein by reference, describes a benchtopanalyser which requires the insertion of disk based cartridgescontaining a predetermined panel of tests. The device is developed foruse of veterinary professionals and its size makes it most useful as abenchtop unit rather than a complete point of care testing device.

WO2013/088429 relates to a method and assay for eliminating the hookeffect in the detection of a target analyte such as an acute phaseprotein in a bodily fluid in which the target analyte comprises a memberof a specific binding pair comprising applying the sample to a solidphase carrier material, generating a signal in accordance withdownstream movement of the labelled first or second members and thetarget analyte to bind with the complimentary immobilised first orsecond members, and detecting the presence of the target analyte inaccordance with the signal generated at the complimentary immobilisedfirst or second members.

DISCLOSURE OF THE INVENTION

The inventors have carried out a number of large scale haematologicaland biochemical studies of racehorses which is unique in the field.

In one aspect of the invention, the inventors have identified specificlevels of SAA which allow fast, accurate assessment of clinical andsubclinical infection, which can be used as a screening tool and allowsmonitoring of disease progression and response to treatment.

WO2013/088429 discusses the use of a particular device for assessingincreased levels of SAA in horses in relation to tissue injury,infection, trauma and arthritis. It further discusses the possible useof SAA to assess reconvalescence of horses recovering from infections orinjury. However WO2013/088429 does not distinguish different possiblecauses of SAA, and nor does it identify particular concentrations orranges which can be used by practitioners to assess infection and takeappropriate action—for example pages 31-32 of WO2013/088429 discuss onlythe severity of “an active inflammatory condition” with the lowest SAAbeing indicative of the ‘mild’ condition being 32.9 μg/ml, and allconcentrations below this being termed “normal”.

However the present inventors have determined that, contrary to theprevailing view in the art, the presence of SAA can be used to actuallydistinguish infection from other types of inflammatory response. Thismeans it can be used alone or in combination with other methods toscreen for and confirm infection, as distinct from other inflammatoryaetiologies, thereby permitting appropriate treatment measures to betaken. They have furthermore characterised the manner in which SAAlevels reflect successful response to treatment of infection. Theseobservations thus provide for improved methods for assessing thesethings.

The inventors further provide novel devices utilising these observationswhich can be used to simply and effectively:

-   -   Confirm with a high degree of confidence whether a subject        exhibiting some abnormal symptom or behaviour is suffering from        an infectious disease, as distinct from some other cause;    -   Rapidly and accurately assess whether a subject being treated        for an infectious disease is responding or not to that        treatment;    -   Screen for the presence of infection, particularly subclinical        infection, in asymptomatic subjects,

In preferred embodiment the device permits visual quantification orsemi-quantification of critical individual concentrations in the rangeof about 15 to 1000 μg/ml (e.g. 15 μg/ml, 50 μg/ml, 200 μg/ml and 1000μg/ml) in the sample, or up to 3000 μg/ml using an electronic reader.The device may include a reference card upon which representations ofthe critical concentrations intensity of are available for comparison.

This device thus allows, inter alia, trainers, breeders andveterinarians to look at their horse's health in a fundamentallydifferent way, enabling them to manage health, not just react toclinical problems.

This research has also identified more generally a need for aninexpensive, portable device for testing levels of exertion indicatorsand other illness indicators in samples of a bodily fluid, particularlyblood, from horses.

The inventors have therefore also developed a system that facilitatesthe collection and then analysis of a blood sample. The system permitsthe generation of an accurately diluted sample solution which can bereadily applied to an assay device by the operator to give a consistentresult rapidly, preferably at the site of sample collection, and issimple to use without specialist knowledge or training.

In a further aspect of the invention, the inventors have identified thatthe combination of CK and AST can be used to manage exertionalrhabdomyolysis, e.g. optionally, but conveniently, these can be measuredusing a system or device described herein which measures both biomarkersat the same time within a suitable concentration range for each.

The device can be used for point of care testing which can be bothperformed and interpreted by the veterinarian and non-veterinarian atthe horse's side.

These and other aspects of the invention will now be considered in moredetail:

Specific Detection of Infection

In a one aspect of the invention, the inventors have identified that SAAlevels allow fast, accurate assessment of clinical and sub-clinicalinfection. Optionally, but conveniently these can be measured using asystem or device described hereinafter.

One of the many components of an inflammatory episode such as can arisefrom infection is an acute phase protein response, in which acute phaseproteins are produced in the liver and released into the bloodstream inresponse to any stimulus causing tissue injury.

Serum Amyloid A is one of the acute phase proteins produced in theliver. Normal levels in healthy horses are very low but increase rapidlyto peak 24 to 48 hours after infection and inflammation (Heegard 2000).

The inventors have determined unexpectedly that point of caredetermination of a critical level of SAA is a reliable marker of equineinfectious inflammation and real time determination (e.g. assessing howquickly highly elevated drop following treatment) may assist greatly inmanagement and monitoring of equine health.

Furthermore it may be useful in screening mammals, for example new-bornmammals, for infection before the onset of symptoms.

Some particular embodiments of this aspect of the invention will now bedescribed in more detail:

Serum Amyloid A (SAA)

SAA is a sensitive and rapid reacting inflammatory protein which can beuseful in screening and monitoring early responses to infection and asubject's response to treatment. Equine SAA is present in threeisoforms, SAA1 and SAA2 and SAA3. SAA1 and SAA2 are both involved in theacute phase response, and reference to “SAA” herein may refer to eitherSAA1 or SAA2, or both SAA1 and SAA2. Each isoform may be detectedindividually, or detected together (i.e. without distinguishing betweenthe different isoforms).

The Equine SAA1 Fasta Sequence is:

LLSFLGEAARGTWDMIRAYNDMREANYIGADKYFHARGNYDAAKRGPGGAWAAKVISDARENFQRFTDRFSFGGSGRGAEDSRADQAANEWG RSGKDPNHFRPHGLPDKYSAA1 FASTA Sequence: http://www.uniprot.org/uniprot/P19857.fastaSAA1 Protein Information: http://www.uniprot.org/uniprot/P19857

The Equine SAA2 Fasta Sequence is:

MKLSIGIIFCSLVLGVSSREWFTFLKEAGQDAWDMWRAYSDMREANYKGADKYFHARGNYDAARRGPGGAWAAKVISDARENAQRVTDLFKFGDSGHGAADSRADQAANEWGRSGKDPNHFRPRGLPDKYSAA2 FASTA Sequence: http://www.uniprot.org/uniprot/F6ZL17.fastaSAA2 Protein Information: http://www.uniprot.org/uniprot/F6ZL17A Tool for Differentiating Between Infectious and Non-InfectiousConditions.

When a horse is presented with clinical/physical symptoms of illness andit is suspected that the cause of the condition is either infectious orindeed non-infectious, testing with SAA may be used to aid in diagnosis.The assessment can be performed based on the likelihood of the potentialaetiology bearing in mind the horse and the environments. In general,this embodiment has particular utility for practicing veterinarians bothin hospitals and ambulatory situations.

As explained in Example 4 the level of SAA was determined in horsesdiagnosed with infectious and non-infectious diseases and was observedto respond most rapidly and dramatically to bacterial and viralinfections, while allergies, EIPH and other non-infectious inflammatoryconditions showed little or no response. SAA levels were also observedto elevate during colic and post-colic surgery, which are both factorsthat can be readily assessed and if need be discounted by those skilledin the art. The case studies compiled in Example 4 demonstrate that SAAis a potent marker of infection and not a marker of generalinflammation, and can thus be used for differentiating betweeninfectious and non-infectious conditions.

SAA was confirmed to elevate in response to some of the most commoninfectious conditions (see Example 4). SAA levels elevate rapidly within24 hours to over 4000 μg/ml in many cases and typically stay elevateduntil the acute phase response is overcome via antibiotic oranti-bacterial treatment or via the body's own immune system.

The benefits of such a method extend to diagnostic procedures where aninfection can be confirmed before further investigation, as well asallowing for the prompt initiation of a suitable treatment regime forsick horses based on whether they are being treated for an infectiousdisease such as those associated with micro-organisms or non-infectiousillness such as those associated with the environment or lifestyle orgenetic factors.

Furthermore SAA has been seen to elevate to a larger extend when thehorse is challenged with a bacterial infection compared to a viralinfection which creates scope for SAA to be used not only as a marker ofdifferentiation between infectious and non-infectious disease but alsoas a method of assisting in differentiating between the organismresponsible which has implications for the type of therapy administerede.g. viral infections will not respond to antibiotic therapy.

In cases where a clinical condition is expected to be non-infectious,SAA may be used to confirm, via negative association, that thatcondition is indeed non-infectious. As shown in Example 4, it has beenconfirmed that SAA does not elevate in response to some of the mostcommon kinds of non-infectious inflammation.

Common Examples in Ambulatory Practice

In racing stables SAA may be used to confirm bacterial lung infectionsafter clinical symptoms such as coughing, snorting or mucus in theairways are observed. Bacterial lung infections in racehorses intraining usually elevate SAA up to 1000 μg/ml. Bacterial lung infectionssecondary to exercise induced pulmonary hemorrhage can be observed 2-3days post strenuous exercise and levels are observed to raise to alesser extent; 30-50 μg/ml.

In sport horse stables such as show-jumping for example, SAA is usuallyused to confirm if a swollen leg or joint is due to bacterial infectionor is a non-septic flare due to some other inflammatory response. Forexample in infected cases SAA is observed to elevate from 1000-5000μg/ml depending on the severity of the infection, while non-septic jointflares do not show any detectable level of SAA. Septic osteoarthritiscan also elevate SAA as high as 1000 μg/ml or above.

Colics both pre- and post-surgery as well as non-surgical cases havebeen shown to cause the elevation of SAA to 1000 μg/ml or higher, evenwhen the horse undergoes surgery and does not receive antibiotics aspart of their post-surgery treatment SAA levels can be observed toreturn to normal within 3-4 days. This is an example of potentiallynon-infectious inflammation where SAA is elevated. Colic however, isvery common and the physical symptoms are readily identified by thoseskilled in the art.

A Tool for Monitoring Progress of Infectious Disease and Response toAnti-Infective/Antibiotic Therapy

As explained in Examples 2 and 3, SAA can be used to efficiently monitorthe recovery of a mammal such as an equine mammal from infection.

In some embodiments a concentration of SAA above about 10, 15, 30, 50,100 or 200 μg/ml indicates the horse should be monitored regularly. Anincreasing concentration of SAA indicates the horse may require medicaltreatment. A decreasing concentration of SAA indicates the horse isimproving, e.g. naturally or in response to medical treatment.

The studies shown in the Examples particularly demonstrate the use ofSAA to determine the biochemical efficiency of the course of treatmentand SAA elevations and decreases were in agreement with clinicalexamination. In particular the data indicated that SAA levels canresolve before the traditional WBC profile returns to normal. Inaddition SAA can be used to determine the efficacy of a treatment bymonitoring the response of the protein post administration.

More specifically, SAA is normally not present (or present only at tracelevels) in equine blood but gets produced in abundance in response toinfectious disease.

The inventors have shown that SAA can elevate from trace levels to 5000μg/ml within 18-24 hours, making it a rapid response diagnostic orprognostic. They have further shown that serum concentration of SAA candrop in response to effective antibiotic or anti-bacterial treatments.The typical pattern in SAA drop-off displays a slight tail on the slope(see FIGS. 17a-c ). In particular the inventors show that the rate ofSAA reduction is an indicator of the effectiveness of the treatment witha fully effective treatment expected to show a half-life reduction ofapprox. 12-24 e.g. 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24hours—more preferably about 16-20 hours, most preferably 17, 18 or 19hours in vivo.

Accordingly, provided herein is a method of diagnosing or monitoring acondition in a horse, comprising

(i) determining the level of SAA in a blood sample from the horse,

(ii) repeating step (i) after a defined period; and

(iii) optionally repeating step (ii),

wherein an increase in the level of SAA indicates the condition isdeteriorating, and a decrease in the level of SAA indicates thecondition is improving.

In some embodiments, the defined period may be about 6, 12, 18, 24, 48or 72 hours.

In some embodiments, the condition is infection, e.g. bacterialinfection or viral infection.

In some embodiments a concentration of SAA above about 10, 15, 30, 50,100, 200 or 1,000 μg/ml indicates the horse should be monitoredcarefully, and/or a treatment step as described herein should be carriedout. Preferably, the treatment step comprises a period of rest for thehorse.

A concentration of SAA below about 10-15 μg/ml, e.g. below about 7.5μg/ml, indicates the horse may be healthy, i.e. have normal performance.However, other markers, e.g. tracheal wash or white blood cell count,may indicate the horse is not yet fully recovered.

A Tool for Screening for Infection in Mammals

In another embodiment of this aspect of the invention, the presentinvention provides novel methods and devices for screening.

In preferred embodiments, a concentration of SAA above about 7.5, 10,15, 20, 25, 30 or 50 μg/ml (micrograms/ml), e.g. above about 10 to 15μg/ml, indicates the potential for infection and impaired horseperformance.

As shown herein, it has been found that horses with an SAA concentrationbetween about 10-15 μg/ml and 200 μg/ml, e.g. between about 15 μg/ml and100 μg/ml, between about 30 and 200 μg/ml or between about 50 and 200μg/ml are unlikely to have visible symptoms. However, their performance,e.g. speed and/or endurance performance, for example in horse racing orother intense exercise, is significantly impaired.

Horses with an SAA concentration greater than about 100 or 200 μg/ml areclinically unwell, e.g. showing clinically significant symptoms orinflammation. Horses with an SAA concentration lower than about 10-15μg/ml, e.g. lower than about 7.5 μg/ml, are considered free fromsubclinical infection, e.g. are expected to perform normally.

Concentrations of SAA above about 10-15 μg/ml indicate increasinglyimpaired performance. In some embodiments, a concentration of SAA aboveabout 30, 50 or 100-μg/ml indicates increasingly impaired performance.

Screening is a process which can conducted “blind” on individuals orgroups of subjects (e.g. horses). There are 3 main utilities forscreening according to the present invention;

-   -   I. Detecting infectious disease        -   a. For disease control        -   b. To identify disease early (sub-clinical)    -   II. Pre-performance testing    -   III. Post-transportation

These will now be discussed in more detail:

I.a. Screening to Detect Infectious Disease for Disease Control

This is a protocol that can be practiced at veterinary inspections, suchas quarantine, ports or borders or prior to inter-state transport in theUS. It may also be conducted to manage disease outbreaks, screen feralpopulations or geographic locations for disease control and/orstatistics.

Assessing SAA according to the present invention has utility as a rapidand simple to use screen or pre-screen to identify potential infectionin the subject.

Common examples of infectious diseases that may be screened orpre-screened in this context are Equine infectious anemia (virus) andPiroplasmosis (protozoa), other equine diseases of lesser interest inthis type of screening are Equine Viral Arteritis and in some casesEquine Herpes Virus types 1 and 4.

In this case the intervention upon a positive result could be to refuseentry of horses into certain event, geographic area or border e.g. astate border.

Methods of the invention may or may not be followed by furtherdiagnostics to confirm the kind of infection. Such confirmatorydiagnostics are well known to those skilled in the art. For example withEIV the commonly used diagnostic is the “coggins test” which is animmunodiffusion test. For Piroplasmosis the confirmatory diagnostic testis typically a PCR.

I.b. Screening to Detect Infectious Disease for Early Identification

This may be conducted as a cost-reduction and/or risk-reduction exerciseat equine facilities such as breeding farms, or competitive trainingfacilities.

Breeding and Rearing Facilities

Common infectious diseases amongst young horses in breeding and raringfacilities are Rhodococcus equi (bacterial infection) and Rotavirus. Aprotocol for effective management of these conditions in neonates wouldbe to screen within 8-10 hours of birth and repeat every 14 days for thefirst 12 weeks of life. R. equi cases were observed to raise SAA as highas 5000 μg/ml within 24 hours while Rotavirus can typically elevate SAAto 1000-2000 μg/ml or higher. Monthly screening would be adequate forolder horses and may be seasonal, e.g. horses could be less susceptibleto these infections in winter.

While there are other markers such as white blood cells and fibrinogenthat elevate in R. equi, Chafflin et. al. (2013) have shown that theyare not reliable for predicting subsequent onset of clinically apparentR. equi (see “Evaluation of Hematologic Screening Methods for PredictingSubsequent Onset of Clinically Apparent Rhodococcus Equi Pneumonia inFoals”. Chafflin M, Coen N, Blodgett G and Syndergaard M. AAEPProceedings. Vol. 59. 2013. p 267; “Evaluation of UltrasonographicScreening Parameters for Predicting Subsequent Onset of ClinicallyApparent Rhodococcus Equi Pneumonia in Foals.” Chafflin M, Coen N,Blodgett G and Syndergaard M. AAEP Proceedings. Vol. 59. 2013. p 268.The present inventors have shown in their unique studies that in R. equiSAA elevates in all tested cases and is a reliable marker when screeningfor R. equi. Furthermore SAA elevates earlier than both WBC andfibrinogen in both R. equi and Rotavirus cases.

If a foal is suspected of R. equi the confirmatory diagnostic isultrasonographic scanning of the lungs. According to Chafflin et. al.(2013) however, 79% of R. equi cases confirmed by ultrasonographicscanning naturally resolve without treatment and therefore it is a poorpredictor of the onset of clinically apparent R. equi.

If R. equi infects a foal it can develop into pneumonia and can causescar tissue formation in the lungs which is detrimental to a racingcareer for example. In circumstances where R. equi has been confirmed,SAA can be monitored to see if the condition is naturally subsiding ornot, whereby SAA is observed to reduce from levels of about 700 μg/ml orhigher to trace levels.

Rotavirus is either confirmed by PCR, or more commonly, the foal isclosely monitored by physical exam for diarrhea (symptom of Rotavirus)and will have its fluids and electrolytes closely managed as well as SAAlevels. Rotavirus can elevate SAA to 1500-2000 μg/ml or greater prior tothe onset of symptoms.

As shown in Example 5, SAA can be used to distinguish between healthynewborns and those who may be seen to develop health problemsimmediately or be susceptible to the development of problems in theweeks and months after birth.

The inventors showed no background SAA count in most subjects. Theinvention can be used to provide an early indicator of future healthproblems, and for the identification of sub-clinical early stageinfection.

Utilities for screening older horses in breeding facilities includesscreening mares for common infections that would cause economic andemotional loss, i.e. losing a pregnancy or newborn. The commoninfections in breeding mares are placentitis (placental infection),bacterial endometritis (uterine infection) and Equine Herpes Virus.

Confirming sub-clinical placentitis is conducted via ultrasound scanningof the uterus and placenta to look for signs of placental thickening,placental/uterine edema, cervical opening, excessive folds or placentalseparation, all of which can be vague. Clinical placentitis would beconfirmed by physical examination for vulvar discharge. Mares withplacentitis may abort or have foals born with complications (which foalscould be screened with SAA upon birth).

Bacterial endometritis would be confirmed via ultrasonographic scanningof the uterus and/or a vaginal swab. The presence of fluid in the uterusand/or bacteria in the swab would indicate the presence of bacterialendometritis, which if present at the time of conception, significantlyincreases the risk of early fetal death.

EHV is confirmed via PCR. EHV causes early abortion and it is routine inlarger breeding facilities to vaccinate against it.

II. Competitive Training Facilities

Common infectious disease in racehorse, trotting horse and endurancehorse training facilities would be unidentified viral infections andbacterial lung infections.

Pre-performance screening is with the aim making a decision to enterinto competition or not, and/or to gamble on the horse winning incompetition. A decision to withdraw from competition may be drawn fromSAA levels greater than 7.5 μg/ml-15 μg/ml, or higher levels (e.g. above100 μg/ml) if it was accepted that a loss of performance might result.

The inventors have showed that 98% of racehorses under 7.5 μg/ml priorto racing performed as expected, while horses with SAA of 15 μg/ml orhigher did not perform as expected.

In these examples, the device is used to make decisions to compete ornot, and/or to estimate the likelihood of winning. Thus in this context,further diagnostics may not be required or conducted. Nevertheless atypical confirmatory diagnostic may be nasopharyngeal endoscopy and/orbronchoalveolar lavage (BAL) to look for traces of bacteria and viruses.Further blood diagnostics may also be conducted such as fibrinogenand/or white blood cells.

Common infections in sport horses, including showjumpers, eventers,endurance horses, dressage horses, quarter horses and others varydepending on geography. In the US the most common infectious diseases ofconcern in these horses are Equine Protozoal Myeloencephalitis (EPM) andLyme disease (bacterial). EPM is usually confirmed via physicalexamination and ELISA based on multiple immunogenic proteins located onthe surface of the parasite. Diagnosis is enabled by determiningserum:CSF titers however testing cerebrospinal fluid is not commonlypracticed due to the collection risk. Lyme disease is also difficult toconfirm and is done so via multiplexed immunoassay which is based on thedetection of antibodies to three B. burgdorferi (causative parasite)antigens in equine serum. The inventors have identified a method ofretrospective diagnosis of EPM and Lyme disease by treating with therelevant treatments and monitoring response to treatment by determiningSAA levels.

III. Post-Transportation Screening

The most common kind of infection post-transportation is pleuropneumoniaa bacterial infection of the lungs. Pleuropneumonia is confirmed byidentifying fluid in the pleura via ultrasound scanning. SAA levels canraise as high as 3500 μg/ml within 24 hours in cases of pleuropneumoniaand stay elevated until the bacterial infection clears, the inventorshave observed that even when fluid resolves in the lungs the acute phaseresponse can stay elevated, therefore SAA provides valuable insight overultra sound scanning alone.

A Broad Range Portable Device for Assessing SAA

The assessment of SAA for the practice of the invention described hereincan be done by any appropriate means known in the art. Preferred bodyfluid collection systems and analytical devices are described by way ofnon-limiting examples hereinafter. Such systems and devices can be usedto measure SAA at both very low and very high levels in the same assay.

In one aspect the invention provides a diagnostic device with an SAAdetection range of 0-3000 ug/ml wherein a result of less than or equalto a specified level (here: about 15 μg/ml e.g. about 7, 8, 9, 10, 11,12, 13, 14 or 15 μg/ml) indicates that there is no infection present.

Preferred devices give a simple indication which is specific to whetherthe concentration of SAA in the body fluid is above or below a specifiedlevel that which has been demonstrated by the present inventors tosuggest that intervention is appropriate.

This can be used in combination with the diagnostically relevant rangeswhich have been identified through haematological and biochemicalstudies and which are described in more detail below e.g. where 7.5-200ug/ml SAA may indicate the presence of subclinical infection and over200 indicates clinical infection which is can be observed onexamination.

Preferred devices are portable and handheld, and can be readily used andunderstood. A preferred device is a portable colour indicator device forthe detection of infectious diseases, which can be used forfield-testing for SAA with immediate results.

In some embodiments, the lateral flow device produces a marker, e.g. acoloured line or stripe which is detectable (e.g. to the naked eye) atan SAA concentration of at or above about 10-15 μg/ml, e.g. about 7.5,10, 12.5 or 15 μg/ml in the body fluid sample. Preferably, the markerincreases in intensity with increased SAA concentration. Preferably, theconcentration of SAA can be reliably determined between a range of about0 and 3,000 μg/ml, e.g. between about 0 and 1,000

Such devices are discussed in more detail hereinafter.

Test Fluid Collection System

Provided herein is a novel test fluid collection system, for collectingand preferably diluting of test fluids.

In preferred embodiments the system is used for body fluids, such asblood, and may be abbreviated herein to “BCS”. However the disclosureapplies mutatis mutandis to the collection of other test fluids—forexample from environmental or industrial sources, or any other source.

As explained in Example 6, the body fluid collection system (preferablyblood collection system) of the present invention simplifies samplecollection and preparation for use preferably with a point-of-carediagnostic test. A typical point-of-care sample handling procedureinvolves collecting the sample (including but not limited to blood,urine or saliva), pipetting a metered volume of sample, adding it to adiluent solution and transferring a measured volume of sample/diluentmixture to the point-of-care device. The system allows for relativelycontactless mixing and distribution of the solution.

The BCS has five main features;

-   -   1. A housing, which includes a fitting portion to engage a        container    -   2. The multi-purpose sample collection tip    -   3. The metered volume sample collection port    -   4. The dispensing inlet    -   5. The dispensing nozzle

The BCS circumvents the use of two different pipettes and reducesvariation introduced by human error. It incorporates a “capillary”action in conjunction with a dispensing nozzle which can be a convenientdropper. When used with the liquid container which provides the diluent,there is formed a ‘mechanical flow path’ whereby reducing the volume ofthe container (e.g. by squeezing) expels the diluted sample through thenozzle.

By combining a sample collection tip, metered volume collection port andmechanical dispensing system, the BCS allows for the collection andpreparation of a sample of blood, for example, without the requirementof blood bottles, blood tubes, pipettes droppers or capillary materials.

While the preferred device is exemplified for blood dilution, the volumeof component to be diluted can be adjusted by the size of the spacing inthe collection port (depth and width) as appropriate to the surfacetension of the liquid to be sampled.

Capillary blood provides a reliable source of biological and/orphysiological information that can be obtained from blood sampling,including but not limited to; blood cell number and morphology,biomarkers, lipids, enzymes, electrolytes, pH as well as other relevantdiagnostic and/or veterinary medical related information.

The body fluid collector may be used in conjunction with a needle,lancet or other suitable blood letting device. It may also be used witha syringe, bottle, tube, blood bottle or blood tube. In one embodiment,the BCS (and in particular the sample collection tip) can be attached toa needle.

Equine capillary blood may be collected from a number of different siteson the equine anatomy, most notably the soft tissue around the gums andmouth as well as the nose and muzzle. Other suitable sites may includethe under-side of the tail, the heels, the sheath or other highlyvascular areas of the anatomy.

Blood or other bodily fluids may be collected by any conventional meansfor use as described herein. Conventional methods for obtaining equineblood samples use the venepuncture method, in which blood is drawndirectly from the equine jugular vein using a syringe or similarapparatus. However, the inventors have determined that a small bloodsample may also easily be obtained for use e.g. with an LFD or otherpoint of care device by puncturing the lip or gum of an equine, forexample using a lancet or similar apparatus.

Thus the body fluid collector enables a body fluid sample (e.g. blood)to be analysed by simply contacting a drop of (e.g.) blood on the body(e.g. gum, lip or skin) of the horse with the body fluid collector. Itcan also collect fluid from the tip of a syringe, bottle or tube or beconnected to a needle to withdraw blood directly from the vein/artery.

In one embodiment the port is optimized for the collection of wholeblood.

In another embodiment the BCS is optimized for the uptake of saliva.

In another embodiment the BCS is optimized for the uptake of serum.

In a further embodiment the BCS is optimized for the uptake of plasma.

In a yet further embodiment the BCS is optimized but not limited to theuptake of any of urine, milk, synovial fluid, peritoneal fluid,cerebrospinal fluid, plant extract, food extract, water or wastewatersamples.

Some particular elements of the system will now be discussed in moredetail:

In one aspect of the invention there is provided a test fluid collectionsystem, for collection of a metered quantity of a test fluid to bediluted for analysis the system comprising a housing comprising a

(i) collection tip

(ii) a collection port

(iii) a dispensing inlet and a

(iv) dispensing nozzle;

wherein the collection tip is at a first end of the housing, and is forcontacting a sample of the test fluid;

wherein the collection port is proximal to and in fluid communicationwith the collection tip and comprises two spaced members having opposinghydrophilic surfaces,

-   -   wherein said surfaces define a volume between them which        corresponds to the metered quantity of test fluid to be        collected;    -   wherein the distance between the spaced members is such that the        test fluid from the sample tip can be drawn into the volume by        capillary action;        wherein the collection tip and dispensing nozzle are at opposite        ends of said housing;        wherein the dispensing inlet is proximal to the collection tip,        and in fluid communication via a dispensing channel to the        dispensing nozzle which runs through said housing;        wherein the housing is adapted to be fitted into the opening of        a liquid container containing liquid for diluting the metered        quantity of the test fluid, with said collection port and said        dispensing inlet within said container, and with a seal fit        between said opening and said housing;        wherein the dispensing nozzle is adapted to dispense diluted        test fluid from the container when the volume of said container        is reduced.        Dimensions

In one embodiment the total longitudinal length of the BCS between 25and 30 mm

In one embodiment the BCS has a circular cross section of circumferencebetween 15 and 20 mm at the widest point.

Collection Tip

The collection tip may be positioned on a collection stem which isconnected to the dispensing inlet and incorporates the collection port.

The collection stem may be elongate to allow for easier samplecollection into the port from (for example) a blood tube—thus is may befor example greater than 20, 25, 30, 40, 45, 50 mm from the collectiontip to the fitting portion of the BCS (described in more detail below).An elongated collection stem may also allow for easier extraction of asample from a tube or bottle.

In a preferred embodiment the BCS allows for pipette-free samplemetering

In another embodiment the BCS allows for venous or arterial blood samplecollection without the requirement of blood tubes or syringes.

Collection Port

As noted above the collection port is composed of two closely alignedflat surfaces

These may be parallel but preferably are not parallel to each other butare slightly angled (e.g. about 1°, 2°, 3°, 4°, 5°, flaring outwardstowards the tip) to encourage greater capillary flow.

In one aspect of the device the surfaces of the collection port arecoated with a hydrophilic coating such as Triton (or any knownhydrophilic material or hydrophilic surface treatment—e.g. a hydrophilicpolymer and/or a polymer coated with a hydrophilic coating.

In one aspect of the device the surfaces of the collection port arecoated with an anticoagulant (e.g. EDTA, Lithium heparin, Sodiumcitrate, or the like).

The corners of the walls or members or the collection port may bebevelled or shaped to increase surface tension in the volume holding thefluid.

The length and width of the space contained within the collection port(between the closely aligned walls) defines the volume that can becollected.

In a preferred embodiment the collection port is composed of two opensided closely aligned walls which form an open channel. In a relatedembodiment the open side walls of the collection port allow the rinsingof blood out of the collection port.

The spacing between the members may be less than 1 mm e.g. less than0.2, 0.3, 0.4, 0.5 mm at its narrowest point spacing.

The width of the members at their widest point may be less than 10 mme.g. about 1, 2, 3, 4, 5, 6, 7, 8, or 9 mm.

The area each opposed surface may e.g. be between 2 and 100 mm².

The distance between the spaced members, and the area of the members,will be selected to enable the desired volume of body fluid sample to bedrawn into to the LFD. Preferably, the collector transfers samplesbetween about 1 and 100 μl, more preferably about 5, 6, 7, 8 8.5, 9, 10,15, 20, 25, 30, 35, 40, 45, 50, 60 70, 80, 90 or 100 μl. Most preferablythe collector collects between about 5 and 30 μl.

Interface with Container

In a preferred embodiment the housing includes a fitting portion whichis adapted to be a push fit into the opening of a container. For examplethis portion may have a cylindrical waist to fit the neck of a bottlevia a push fit where it can be retained by friction or compression. Thefitting portion size is adapted to fit and create a seal with anystandard commercially available bottle.

It may include a frusto-conical portion which is tapered (inwardlytowards the collection tip). This permits easy insertion of the fittingportion into e.g. a bottle neck.

In one embodiment of the BCS the fitting portion is cylinder (optionallytapered) of at least 5, 6, 7, 8, 9 or 10 mm in longitudinal length. Itmay be less than 20, 19, 18, 17, 16, 15 mm in length.

In one embodiment the diameter of the fitting portion is adapted to fitwithin a bottle or container with an inner neck diameter of 5 mm, 6 mm,7 mm, 8 mm, 9 mm, 10 mm, 11 mm, 12 mm, 13 mm, 14 mm, 15 mm, 16 mm, 17mm, 18 mm, 19 mm, 20 mm, 21 mm, 22 mm, 23 mm, 24 mm 25 mm, 26 mm, 27 mm,28 mm, 29 mm, 30 mm, 31 mm, 32 mm, 33 mm, 34 mm, 35 mm.

Thus in one embodiment the diameter of the fitting portion is 1 mm, 2mm, 3 mm, 4 mm OR 5 mm wider than the inner dimension of the neck of theaccompanying bottle in order to create an appropriate seal, asappropriate to the resilience of the material of the container neck andfitting portion.

A preferred diameter of the fitting portion is between 15 and 20 mm e.g.16, 17, 18, 19 mm.

A most preferred embodiment has a circumference of 18.7 mm which fitsconveniently into a bottle with an internal bottle neck diameter of 14mm.

In a preferred embodiment the BCS system is used in conjunction with aplastic (compressible) bottle made from a low-density polyethylene(LDPE).

In another embodiment the BCS allows for pipette-free sample preparation

Dispensing Diluted Fluid

Generally inversion of the container (e.g. bottle) to which the BCS canbe attached can result in the entry of diluted sample into the dispenseport and along the channel where it travels to the dispense nozzle.

Preferably pressure to the container forces diluted sample through thedispense nozzle where it is formed into measured droplets according tothe inner dimensions and slope (as mentioned below) of the dispensernozzle.

Preferably the dispense nozzle is dimensioned to determine and controldrop size (e.g. 10-300 μl)

The dispense channel may be flared at the dispense nozzle exit whichaids in the creation of a uniform drop.

An elongated dispense nozzle may also allow for easier mixing as it isimmediately immersed in the diluent on attachment of the BCS to thebottle

In a preferred aspect of the device the sample collection and dispenseactions of the BCS are separate functions which can be performedindependently of each other.

In another embodiment the BCS allows for metering and dispensing in thesingle device without mechanical or moving parts.

Construction

The BCS may be made of transparent materials thereby giving feedback tothe user when the collection port is filled and\or dispensing isoccurring.

Preferably, the surface of the mold used to make the channel walls willhave a high polish finish (SPI A1 finish), and may optionally haveadditional hard chrome plating.

A preferred BCS device is described in the Examples and Figureshereinafter.

Analytes for Indicating Equine Exertional Rhabdomyolysis (EER)

Equine exertional rhabdomyolysis (ER) is a debilitating condition thatoccurs mainly as a response to exertion, resulting in varying degrees ofstiffness and pain due to muscle damage (Landau et al. 2012). Sporadiccases can occur when a horse exercises at a level above its fitnesswhile chronic cases are often attributable to an underlying heritablecondition. Equine creatine kinase (CK) levels spike within 4-6 hours ofER and return to normal within 3 days to 7 days (EL-Deeb, 2012).Aspartate aminotransferase (AST) activity peaks approximately 24 hoursafter an episode of ER and may require several days to weeks to returnto basal levels (Cardinet, 1997).

If a horse experiences an episode of ER during exercise it will set thehorse back in its training program causing significant economic loss.Conversely if a horse unnecessarily foregoes exercise as a precautionfor ER, its training schedule will also be compromised. The inventorshave determined that having a real-time diagnosis of CK and AST levelscan aid in the predicting the onset of ER and can be used to determineif a horse has tied up, the severity of the episode and when the horseis recovering. In the study described in the Examples below, 26% of thehorses in the study tied up over a 6 month period and of those 14recurrent episodes were recorded, illustrating the severity of theproblem. The present invention can assist greatly in managing ER and theresponse to treatment, which for example may involve careful nutritionalmanagement.

Aspartate Aminotransferase (AST) and Creatine Kinase (CK)

AST is present in two isoforms in equines, encoded by GOT1 and GOT2respectively. Elevations of AST are seen in the presence of myopathy orhepatopathy. After muscle damage, AST levels peak at 24-48 hours andgenerally return to baseline concentrations within 10-21 days assumingthat no further damage occurs. An elevation in the muscle isoform of CKis specifically seen in acute phase myopathy. CK levels peak at 6-12hours and return to baseline levels within 3-4 days.

Equine AST FASTA Sequence

MTSPSIFVEVPQAQPVLVFKLTADFREDPDPRKVNLGVGAYRTDDCQPWVLPVVRKVEQKIANNSSLNHEYLPILGLAEFRSCASRLALGDDSPALQEKRVGGVQSLGGTGALRIGAEFLSRWYNGTNNKNTPVYVSSPTWENHNGVFSGAGFKDIRSYHYWDATKRGLDLQGFLNDLENAPEFSIFVLHACAHNPTGTDPTPEQWKQIASVMKRRFLFPFFDSAYQGFASGNLDRDAWAVRYFVSEGFELFCAQSFSKNFGLYNERVGNLTVVAKEPDSILRVLSQMQKIVRITWSNPPAQGARIVAFTLSDPGLFKEWTGNVKTMADRILSMRSELRARLEALKTPGTWNHITEQIGMFSFTGLNPKQVEYLVNQKHIYLLPSGRINMCGLTTKNLDYVATSIHEAVTKFQGOT 1 FASTA Sequence: http://www.uniprot.org/uniprot/P08906.fasProtein Information: http://www.uniprot.org/uniprot/P08906Equine Creatine Kinase FASTA Sequence, CKM

MPFGNTHNKFKLNYKPEEEYPDLSKHNNHMAKALTFDIYKKLRDKETPSGFTLDDVIQTGVDNPGHPFIMTVGCVAGDEESYVVFKELFDPIIQDRHGGYKPTDKHKTDLNHENLKGGDDLDPHYVLSSRVRTGRSIKGYTLPPHCSRGERRAVEKLSVEALNSLTGEFKGKYYPLKSMTEQEQQQLIDDHFLFDKPVSPLLLASGMARDWPDARGIWHNDNKSFLVWVNEEDHLRVISMEKGGNMKEVFRRFCVGLQKIEEIFKKAGHPFMWNEHLGYVLTCPSNLGTGLRGGVHVKLAHLSKHPKFEEILKRLRLQKRGTGGVDTAAVGSVFDVSNADRLGSSEVEQVQLVVDGVKLMVEMEKKLE KGQSIDDMIPAQKhttp://www.uniprot.org/uniprot/F7BR99.fastahttp://www.uniprot.org/uniprot/F7BR99

When both CK and AST (which takes longer to rise, peak and return tonormal) are measured, the inventors the inventors have determined thatEER can be accurately diagnosed. Further, the response to treatment canbe usefully monitored.

Levels of AST and CK for Use in Management of Lying Up′

Normal concentrations of CK differ from horse to horse and betweendifferent veterinary clinics, but blood levels below 200 units/l aregenerally considered normal.

The large study carried out by the inventors has shown that AST can beused as a late marker of ER at an activity level of 1000 units/l.

Further, the inventors have determined that a concentration of CK of 500units/l in sport horses is acceptable and tying up is indicated atconcentrations of 700 or 800 units/l and above. Horses should thereforebe carefully monitored if levels rise above 500 units/l.

As such provided herein is a method of monitoring the presence of,severity of, progression of, or recovery from, equine exertionalrhabdomyolysis in a horse, the method comprising

(i) determining the level of CK and AST activity in a blood sample overa period of time;

(ii) determining whether:

the activity level of CK is above or below 200, e.g. about 300, 400,500, 600, 700, or 800 units/ml and/or

the activity of AST is above or below about 1000 units/ml, wherein

an activity level of CK:

of about 700-900 units/ml indicates risk of onset of equine exertionalrhabdomyolysis, and of about 900-1100 units/ml indicates onset of equineexertional rhabdomyolysis, accompanied by an activity level of AST aboveor below 1000 units/ml indicates equine exertional rhabdomyolysis.

In one aspect the method comprises repeating the determination atintervals, whereby a reduction of the CK to below 500 from above 500, ora reduction of AST below 1000, indicates recovery from equine exertionalrhabdomyolysis.

In one embodiment, an indication of equine exertional rhabdomyolysis, orthe onset or risk of equine exertional rhabdomyolysis is followed by atreatment step. For example, the treatment step may comprising a periodof rest for the horse.

In one embodiment, a determination of exertional rhabdomyolysis, or theonset or risk of equine exertional rhabdomyolysis is followed byexamination of the training regime and/or nutritional plan of the horseto determine if any changes have been made which may have added to theonset of the episode.

In another embodiment the device is used to determine baseline CK and/orAST levels before a change in a training regime is implemented. CKand/or AST levels are then monitored after the change has beenimplemented to determine the effect on CK and/or AST levels and toassess the risk of the onset of an episode of exertional rhabdomyolysise.g. in the event that CK rising to about 500-600 units/ml wasdetermined.

In a further embodiment the device is used to determine baseline CKand/or AST levels before a change in the nutritional plan of a horse isimplemented. CK and/or AST levels are then monitored after thenutritional change has been implemented to determine the effect on CKand/or AST levels and to assess the risk of the onset of an episode ofexertional rhabdomyolysis e.g. in the event that CK rising to about500-600 units/ml was determined.

In another embodiment CK/AST levels are monitored along with a trainingregime and/or nutritional plan to develop an exertional rhabdomyolysismanagement system whereby baseline levels of CK and/or AST are reduced.

In a preferred aspect of the invention the analysis of the twobiomarkers is combined in a single device e.g. which is portable andheld-held and indicates the concentration bands described above. Exampledevices for measuring two biomarkers such as CK and AST are described inmore detail below.

Lateral Flow Devices and Other Preferred Embodiments

The analysis of any one of SAA, fibrinogen, CK and\or AST (or otheranalyte of interest provided through the use of the blood collectionsystem) may be performed using any analytical device known in the art.

Described herein by way of non-limiting example is a lateral flow device(LFD) that can be applied to the measurement of levels of any or all theabove indicators in bodily fluids, in particular to levels in blood.

In one embodiment the LFD provided herein incorporates a bloodcollection device which draws a blood sample into the LFD for subsequentanalysis.

In other embodiments an LFD which does not incorporate a bloodcollection device can be used in conjunction with the BCS to assay otherindicators in blood, for example IgG and other immune markers.

The LFD and the reader provided herein are, in preferred embodiments,inexpensive to produce and costs to the user are also reduced by thefact that there is no need for the samples to be sent to a laboratory.This, combined with the ease of use of the system, means that samplescan be analysed much more frequently and quickly than is currentlypossible, which may be of great value.

A lateral flow assay device for the analysis of body fluid may thuscomprise:

(i) a housing, and

(ii) a flow path, and (iii) optionally, a body fluid collector, whichforms an integral part of the flow path.

As used anywhere herein, unless context demands otherwise, the term‘body fluid’ may be taken to mean any fluid found in the body of which asample can be taken for analysis. Examples of body fluids suitable foruse in the present invention include, but are not limited to blood,urine, sweat and saliva. Preferably, the body fluid is blood. Asdescribed herein, the fluid may be diluted by a pre-determined amountprior to assay, and any quantification indicator on the LFD may reflectthat pre-determined dilution.

Preferably, the devices, systems and methods described herein are formeasuring analyte levels in equines such as horses (Equus feruscaballus), and find particular use with racehorses for monitoring healthand anticipating performance, for sport horses, racehorses and foals formonitoring disease progression and response to treatment, and fornewborn foals—as an infectious disease screening method. However, theskilled reader will appreciate that the embodiments described herein canequally be adapted for other animals, especially mammals includinghumans.

An integrated body fluid collector, where present on the LFD, may becontinuous or contiguous with the flow path, allowing body fluid (e.g.blood) to be wicked directly from the subject (e.g.) horse into thelateral flow device by capillary action. In other words, the body fluidcollector enables a body fluid sample (e.g. blood) to be analysed bysimply contacting a drop of (e.g.) blood on the body (e.g. gum, lip orskin) of the horse with the body fluid collector. By contrast, manylateral flow devices are designed to receive a sample by pipetting itonto a sample port.

Further, the lateral flow devices described herein may be are capable ofoperating in any orientation. By contrast, most commercial assaysrequire a flat surface for test operation.

Some aspects of the device will now be discussed in more detail:

Flow Path of LFD

The flow path (e.g. a chromatographic strip) is preferably provided by acarrier, through which the test substance or body fluid can flow bycapillary action. In one embodiment, the carrier is a porous carrier,for example a nitrocellulose or nylon membrane. In a further embodiment,sections or all of the carrier may be non-porous. For example, thenon-porous carrier may comprise areas of perpendicular projections(micropillars) around which lateral capillary flow is achieved, asdescribed in for example WO2003/103835, WO2005/089082 and WO2006/137785,incorporated herein by reference.

The flow path will typically have an analyte-detection zone comprising aconjugate release zone and a detection zone where a visible signalreveals the presence (or absence) of the analyte of interest. The testsubstance can be introduced into the LFD by direct contact with themouth of the body fluid collector, and flows through to the detectionzone.

Preferably the carrier material is in the form of a strip, sheet orsimilar to the material described in WO2006/137785 to which the reagentsare applied in spatially distinct zones. The body fluid sample isallowed to permeate through the sheet, strip or other material from oneside or end to another.

Analyte Detection Methods

Analyte detection may be based on competitive or sandwich(non-competitive) assays. Such assays may be used to detect, SAA, or CKand AST, as described herein.

The conjugate release zone contains freely mobile specific bindingpartners to the analyte of interest. For example, if the analyte is anantigen, its binding partner may be an antibody. For example, theantibody may bind any one of SAA, fibrinogen, CK or AST. Alternatively,the conjugate release zone may comprise reagents for carrying out aparticular assay to enable detection of the analyte, as describedherein.

The binding partners may be attached to a mobile and visible label. A“label” is a composition detectable by spectroscopic, photochemical,biochemical, immunochemical, electrical, optical or chemical means.Useful labels in the present invention include magnetic beads (e.g.,Dynabeads™), fluorescent dyes, radiolabels, enzymes, and colorimetriclabels such as colloidal gold, silver, selenium, or other metals, orcoloured glass or plastic (e.g., polystyrene, polypropylene, latex,etc.) beads. Preferred is a gold colloid or latex bead.

If the analyte is present in the sample, it will bind to the labelledbinding partners. In preferred embodiments the intensity of the colourmay be directly proportional to the amount of analyte. Here thedetection zone comprises permanently immobilised unlabelled specificbinding reagent for the same analyte. The relative positioning of thelabelled binding partner and detection zone being such that a body fluidsample applied to the device can pick up labelled binding partner andthereafter permeate into the detection zone. The amount of bound labelcan be detected as a visible signal in the detection zone.

The label in the LFD will be quantifiable by conventional means or asdescribed herein.

In one competitive format embodiment, the detection zone containsregions of immobile analyte-protein derivatives. These bind andimmobilise any of the labelled binding partners not already bound by theanalyte in the sample, producing a coloured line or stripe. In this casethe amount of label bound in the detection zone (and hence the intensityof the coloured stripe) will be inversely proportional to the amount ofanalyte in the sample.

In another competitive format, a labelled analyte or analyte analoguemay alternatively be provided and this is detected using immobilizedspecific binding partner (e. g. immobilized antibody specific for theanalyte) in the detection zone.

In another competitive format, a labelled analyte or analyte analogue isprovided along with a specific binding partner (e.g. an antibodyspecific for the analyte). The resulting mixture is conveyed to thedetection zone presenting immobilized binding partner of the analyte oranalyte analogue. The higher the amount of analyte in the sample, thehigher the amount of free labelled analyte which leaves the conjugaterelease zone to be detected in the detection zone.

In one LFD which may be used, the flow path has:

(a) optionally, a body fluid collector for transferring a sample of thebody fluid into the flow path by capillary action;

(b) optionally, a blood filter, integrated into the body fluid collectoror positioned in the flow path downstream of the body fluid collector;

(c) a carrier along which the body fluid is capable of flowing bycapillary action, wherein the carrier is positioned in the flow pathdownstream of the body fluid collector, the carrier comprising:

-   -   (i) an analyte-detecting means, capable of providing an assay        result indicative of the presence of the analyte; and    -   (ii) optionally a control zone, positioned on the carrier        upstream or downstream of the analyte-detecting means, capable        of indicating the assay has been successfully run;        Control Zone

Preferably the LFD for use with the present invention contains a controlzone, which may be located after the detection zone in the direction ofsample flow, in which excess labelled binding partner binds to produce avisible signal showing that the test has been successfully run.

Alternatively or additionally, a control zone may be located before thedetection zone in the direction of sample flow, indicating that enoughsample has been collected to allow operation of the test.

In one embodiment, the control zone is used as a reference point for areader (see below).

In an example of another control zone, control reagents could be chosenwhich display similar characteristics to the analyte (e.g. SAA) testline in terms of time to appear.

Integral Body Fluid Collector

Where the LFD integrates its own body fluid collector, this may have ahousing made of any material, and the capillary channel is locatedwithin this housing. Preferably, the housing is transparent or partiallytransparent. This enables user feedback during sample collection,enabling the user to determine if body fluid has been drawn into theLFD. In one embodiment, the body fluid collector housing is integratedwith the housing of the LFD.

In one embodiment, the body fluid collector may have a mouth ispositioned in a flat wall in the housing of the LFD and a channel toenable the desired volume of fluid to be drawn into the LFD.

Preferably, the mouth of the channel is between about 2 and 30 mm wideand between about 1 and 10 mm high. Preferably the mouth issubstantially rectangular. In one embodiment, the mouth is about 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29 or 30 mm wide. The mouth may be about 1, 2,3, 3.5, 4, 5, 6, 7, 8, 9 or 10 mm high. The capillary mouth therebyallows rapid collection in case the horse moves during samplecollection. In one preferred embodiment, the mouth is between about 5and 15 mm wide and 2 and 8 mm high. In one embodiment the mouth is 8 mmwide and 0.5 mm high.

Further, the broad opening allows the device to come into contact with alarger area of tissue. The broad opening also transports the sampleacross a large surface area allowing for a faster wicking rate from thetissue surface. The broad opening tapering to narrow exit furtherenhance capillary force, altogether reducing sample exposure time andimproving reliability and consistency. These points combined increasecapillary flow, reduce sample exposure time and improve reliability andconsistency.

The body fluid collector may collect a metered or unmetered volume ofblood.

An integral collector may have any of the structural characteristics ofthe BCS described above, in terms of channel and wall alignment, anddimensions and structures.

Two or More Analytes

In various aspects of the invention, the LFD may be capable of detectingtwo (or more) different analytes e.g. CK and AST.

In one embodiment, the two analytes are analysed using two distinct flowpaths. Preferably, each flow path will have a distinct body fluidcollectors. Preferably, the housing of the LFD houses the two flowpaths.

For example, the flow path may comprise two or more carriers. Thecarriers may be positioned along the flow path consecutively. In use,body fluid would flow along each carrier sequentially.

In a further embodiment, two or more carriers may be positioned in theflow path in parallel. In use, body fluid would flow along each carriersimultaneously. Each carrier may still be in fluid connection with asingle body fluid collector.

In a further embodiment, the lateral flow device may comprise two flowpaths.

In one embodiment, the analyte-detecting means may comprise a firstbinding reagent that specifically binds the analyte and a second bindingreagent that specifically binds the analyte, wherein the first bindingreagent is labelled and is movable through a carrier under the influenceof a liquid by capillary flow and the second binding reagent isimmobilised at a detection site in the flow path. The analyte-detectingmeans comprises a labelled, mobile antibody, specific for the analyteand an immobilised unlabelled antibody, specific for the analyte.

In one embodiment, the analyte-detecting means for each analyte may bepositioned together on the carrier, but the specific analyte-bindingreagent for each different analyte may comprise a different label. Thedifferent labels will be capable of being distinguished as describedherein or by conventional means.

Alternatively, the analyte-detecting means for each analyte may bespatially distinct. The flow path in the ‘multiplexed’ LFD mayincorporate two or more discrete carriers of porous or non-porous solidphase material, e.g. each carrying mobile and immobilised reagents.These discrete bodies can be arranged in parallel, for example, suchthat a single application of body fluid sample to the device initiatessample flow in the discrete bodies simultaneously. The separateanalytical results that can be determined in this way can be used ascontrol results, or if different reagents are used on the differentcarriers, the simultaneous determination of a plurality of analytes in asingle sample can be made. Alternatively, multiple samples can beapplied individually to an array of carriers and analysedsimultaneously.

Preferably, multiple analyte detection zones may be applied as linesspanning or substantially spanning the width of a test strip or sheet,preferably followed or preceded by one or more control zones in thedirection of body fluid travel. However, multiple analyte detectionzones may also, for example, be provided as spots, preferably as aseries of discrete spots across the width of a test strip or sheet atthe same height. In this case, a one or more control zones may again beprovided after or before the analyte detection zones in the direction ofbody fluid travel.

Preferred Combined CK and AST Device

In one aspect of the invention there is provided an LFD device formonitoring the presence of, severity of, progression of, or recoveryfrom, equine exertional rhabdomyolysis in a horse, which devicecomprises means for determining whether levels of CK are above or below200 units/l or in excess of 300 units/l or 400 units/l or 500 units/l or600 units/l or 700 units/l or 800 units/l, up to 3500 units/l and/orwhether levels of AST are above or below about 1000-3500 units/l.

As explained in Example 7, a preferred LFD has

(i) a housing having a sample port, and

(ii) at least one flow path or channel which includes a carrier throughwhich the test substance or body fluid can flow from the sample port bycapillary action,

-   -   wherein the flow path has:    -   an analyte-detection zone comprising a conjugate release zone        and a detection zone where a visible signal reveals the presence        of each analyte of interest.    -   optionally a control zone, positioned on the carrier upstream or        downstream of the analyte-detecting means, capable of indicating        the assay has been successfully run;

The flow path may comprise two or more carriers. The carriers may bepositioned along the flow path consecutively. In use, body fluid wouldflow along each carrier sequentially.

In a further embodiment, two or more carriers may be positioned in theflow path in parallel. In use, body fluid would flow along each carriersimultaneously. Each carrier may still be in fluid connection with asingle sample port.

In a further embodiment, the lateral flow device may comprise two flowpaths.

In one embodiment CK and AST concentrations in particular are determinedby colourimetric enzymatic means on a chromatographic strip.

In one embodiment, the result of the determination is indicated by thepresence and intensity or absence of a coloured test line for CK andAST. The presence and intensity of the coloured test line can be used tomonitor the increase or decrease of CK and AST and therefore the onsetof and recovery from exertional rhabdomyoysis. The housing may comprisean indication of which test line corresponds to which analyte.

LFD Reader

When using LFDs in performance of the present invention, the intensityof the signal in the detection zone may be converted to a quantitativereading of the concentration of analyte in the sample. It is thereforepreferred that the LFD can be used in conjunction with a screeningdevice (reader). The reader is preferably a handheld electronic deviceinto which the LFD cartridge can be inserted after the sample has beenapplied.

The reader comprises a light source such as an LED, light from whichilluminates the LFD membrane. The reflected image of the membrane may bedetected and digitised, then analysed by a CPU and converted to a resultwhich can be displayed on an LCD screen or other display technology (oroutput via a conventional interface to further storage or analyticalmeans). A light-dependent resistor, phototransistor, photodiode, CCD orother photo sensor may be used to measure the amount of reflected light.The result may be displayed as positive or negative for a particularanalyte of interest or, preferably, the concentration of the particularanalyte may be displayed. More specifically the conventional readercomprises: illuminating means for illuminating an immunoassay test;photosensitive detector means for detecting the intensity of light fromthe illuminating means which is reflected from the immunoassay test;means, coupled to the output of the photosensitive detector means, forrepresenting the intensity of the detected light by a data array; memorymeans for storing preset data; first data processing means, coupled tothe memory means and to the output of the means for representing theintensity of the detected light by a data array, for segmenting the dataarray according to the preset data into control data, background dataand test data; second data processing means, coupled to the first dataprocessing means, for determining whether the test data exhibits astatistically significant result; and output means, coupled to theoutput of the second data processing means, for outputting the resultsfrom the second data processing means.

In embodiments of the present invention where multiple analytes areassessed, the reader may analyse the results to detect a plurality ofspatially distinct detection or test zones pertaining to differentanalytes. The photosensitive detector means (e.g. light dependentresistor, phototransistor, photodiode, CCD or other light sensor) willtherefore detect reflected light from all of these (optionally scanningthem) and generate a discrete or segmented data stream for each zone.Respective control zonal data and background zonal data may also begathered for the different analytes.

The colour of the LED or other source may vary dependent on the label ormethod of detecting the analyte.

For gold-labelled analytes, a white LED may be preferable, and thereforea reader may comprise both a red and white LED.

Other Detection Systems

Alternatively, the intensity of the signal in the detection zone may bedetermined by eye, for example by comparison to a reference chart orcard. Provided herein is a reference card which enables theconcentration of the analyte or analytes, e.g. SAA, to be determined bycomparison of the signal intensity in the detection to the referencecard. Preferably, the reference card displays the signal intensity forthe ranges of SAA described herein, e.g. 15, 50, 200 and 1000 μg/ml.Preferably, the reference card shows a control line signal intensity,i.e. the visible signal showing that the test has been successfully run.An example reference card is shown in FIG. 13.

One aspect of the invention provides a kit comprising an LFD of theinvention as described herein and a reference card as described herein.

Other Components

In a further embodiment the LFD comprises a blister pack or pouchcontaining a buffer. The buffer may be released, for example bycompressing an indicated area of the LFD, following sample application.The released buffer encourages sample flow through the flow path, asknown in the art. Alternatively, a buffer may be added manually, forexample by pipette.

In embodiments where a buffer is not used in a pouch/blister pack oradded manually by pipette, then the flow path may contain reagents in adried form which, when wetted, aid flow or generally improve theperformance of the test. Such reagents may be positioned for example inor downstream of the body fluid collector, or in the carrier, forexample in the conjugate release zone. Such reagents may comprise Tween20, PEG, Polyvinylpyrrolidone (PVP), BSA, or a combination of theseand/or other known detergents.

In some embodiments, a hydrophilic tape such as ARflow® 90128, describedin WO 02/085185 A2 (incorporated by reference herein) is incorporatedinto the flow path to further encourage flow.

FIGURES

FIG. 1: Lateral flow assay device with integrated body fluid collector

FIG. 2: Lateral flow assay device with integrated body fluid collector

FIG. 3: Lateral flow assay device with a traffic light system of go,caution and stop

FIG. 4: Correlation of elevated fibrinogen and elevated SAA to eachother

FIG. 5: Relationship between elevated Fibrinogen, elevated SAA andabnormal total white blood cell count

FIG. 6: Elevated Fibrinogen and Total White Blood Cell Count Correlation

FIG. 7: Highly Elevated Fibrinogen (>5 mg/ml) and Total White Blood CellCount Correlation

FIG. 8: Correlation between normal fibrinogen and low total white bloodcells

FIG. 9: Correlation between normal fibrinogen and high total white bloodcells

FIG. 10: Relationship between elevated SAA and normal total white bloodcells

FIG. 11: Correlation between SAA levels and horses which performed asexpected and those that did not. All but one horse performed as expectedwhen SAA was measured at 7.5 μg/ml or below.

FIG. 12: SAA concentrations during the recovery of a horse diagnosedwith travel sickness.

FIG. 13: Example reference card for determining SAA levels.

FIG. 14: Test fluid collection system according to the presentinvention.

FIG. 15: Bar chart showing recurrence of “tying up” in horse populationtested in Example 7.

FIG. 16: Examples of devices for monitoring “tying up” by assay of CKand AST.

FIG. 17: SAA concentrations determined in horses during infection,compared with other indicators. This demonstrates the significantutility of SAA as a prognostic or diagnostic marker.

The invention is described herein by way of example and not limitation,by reference to the accompanying drawings. Many equivalent modificationsand variations will be apparent to those skilled in the art when giventhis disclosure. Accordingly, the exemplary embodiments of the inventionset forth are considered to be illustrative and not limiting. Variouschanges to the described embodiments may be made without departing fromthe spirit and scope of the invention. All documents cited herein areexpressly incorporated by reference.

EXAMPLES Example 1

1. Introduction

Racing thoroughbred horses have been selectively bred to produce optimalperformances of speed and endurance on the race track. In order toachieve athletic excellence the horse must undergo a rigorous exerciseprogramme. Just as human athletes strive to find the right balancebetween training hard enough to maximise performance but not so hardthat stress induces either injury or a compromised immune system, so toowith the horse trainers [1]. Since clinical symptoms in horses may onlyappear when over-stressing has already occurred, methods to determineimminent problems at sub-clinical stages are at a premium. Currentmethods of detecting when health is becoming compromised focus on bloodbiomarkers. Of three current measures, red blood cell counts, whiteblood cell counts and blood biochemistry, the most commonly used istotal white cell count, usually coupled with estimates of the relativeabundance of the five main types of white cell, the neutrophils,lymphocytes, monocytes, eosinophils and basophils. White cell counts canchange rapidly in response to adverse health but the changes tend to betransient and to differ depending on the stimuli. For example the totalwhite cell count may decrease to below normal in response to acuteinflammation or virus attack but may increase in response to prolongedinflammation or bacterial infection [2]. Similarly, neutrophils, whichnormally make up 60% of the total white cells, may decrease quickly inresponse to acute stress but increase quickly when fighting acuteinfection [3]. Nonetheless, neutrophil and lymphocyte counts can be usedto diagnose airway inflammation disease and recurrent airway obstruction[4] using bronchoalveolar lavage.

Although the various white cell counts have the potential to indicate arange of common conditions, there are a number of important issues.First, and most importantly, changes in white cell numbers can occur forreasons other than disease or injury, such as being agitated at the timeof blood collection. Second, base levels are rather variable, withyounger thoroughbreds in particular differing greatly in their whitecell counts from one week to another without any evidence of infectionor inflammation [5]. Third, the fact that cell number can go down aswell as up may cloud the interpretation of tests where multiple opposingstimuli are present. For these reasons, trainers often treat white bloodcells with skepticism as being too difficult to understand and toovariable to provide a reliable indicator of a horse's overall healthprofile.

A more reliable tool should aim to reflect specifically the changes inblood biochemistry that occur at the onset of stress. When an animalsuffers tissue injury, acute phase proteins are produced in the liverand released into the bloodstream and the result is localisedinflammation. Similar responses are noted for a wide range of conditionsincluding trauma, arthritis, surgery or bacterial, viral and parasiticinfection [6,7,8] indicating that the acute phase response is genericand may be mounted to any form of tissue damage. Acute phase proteinsthus appear a logical target for an improved test for stress-relatedinjury during training. Two promising candidate proteins are fibrinogen,which has been the most commonly measured acute phase protein for sometime, and serum amyloid A (SAA), which is becoming increasingly popularas a diagnostic of acute infection.

Fibrinogen is a plasma glycoprotein synthesised by the liver and isconverted by thrombin into fibrin during blood coagulation. Fibrinogenis normally present at between 2-4 mg/ml but this rises followinginflammation regardless of the cause. Indeed, fibrinogen may be the soleindicator of inflammation [9, 10, 11,]. Elevated levels of fibrinogenmay indicate chronic inflammation or reflect the progression of aninfection [12]. Novel inflammation causes the level of fibrinogen toincrease above normal within 24-48 hours and in proportion to the degreeof inflammation, and remain elevated for up to 10 days [13]. Thisrelatively rapid response means that fibrinogen elevation may occurbefore clinical symptoms of illness [14, 15].

Serum Amyloid A (SAA) is a second acute phase protein that is alsoproduced in the liver. Normal levels in healthy horses are very low butincrease rapidly to peak 24-48 hours after infection [16]. CirculatingSAA concentrations may increase up to 100 fold in response to aninfection [13] but it disappears rapidly after the infection has abated[17], making it an excellent ‘real time’ diagnostic tool for trackingprogression and recovery. Previous studies have shown that elevated SAAmay also be used for detecting the presence of inflammatory disease ofthe airways [6], gut [18] and musculoskeletal system [7, 19]. As withfibrinogen, the severity of the inflammation is reflected in the degreeof elevation of SAA.

The purpose of the current study is to investigate the relationshipbetween classic white cell counts and the two indicators of aninflammatory response, fibrinogen and SAA across a large sample ofthoroughbred horses in training. We find evidence that WBC, fibrinogenand SAA capture different aspects of a horse's physiology. WBC countsfluctuate across a rather narrow range and correlate well with parallelchanges in many elements of blood chemistry, suggesting that they tracknormal homeostatic fluctuation. In contrast, fibrinogen and SAA tend tovary little except in a small subset of horses where both markers tendto show markedly elevated levels.

2. Materials and Methods

A population of thoroughbred horses bred for flat racing were screenedat two random dates, once at the beginning of the racing season (1-2 May'12 n=105) and once at the end of the racing season (2-3 Sep. '12n=118). The horses were a random mixture of males and females, a mixtureof grades, ranging in age from 2 to 5 year old and had raced a maximumof 5 times each. All horses are managed in the same way with individualboxes, photoperiod of 4:30 am to 9 pm, a natural indoor temperature (18to 20° C.) and the same feeding and training schedules. The horsesunderwent one workout of approximately 20-30 minutes per day between thehours of 6 am and 10 am. The horses were allowed to rest for a period of4-7 hours post exercise before blood draw. Detailed veterinary analysisof each horse immediately post sampling would be desirable but wasbeyond the scope of the current study. The horses names, existinginjuries, illnesses and medications were not recorded, however it wasnoted by the veterinarian that all horses were fit for work. A largedegree of overlap between the 2 sets of horses tested is expected. Thecomplete blood count consists of the red cell series (Red Blood Cell(RBC) Count, Hemoglobin (Hgb), Hematocrit (Hct), Mean Corpuscular Volume(MCV), Mean Corpuscular Hemoglobin (MCH), Mean Corpuscular HemoglobinConcentration (MCHC), platelets (Plt) and the white cell series (TotalWhite Blood Cells, Neutrophils (Neut), Lymphocytes (Lymph), Monocytes(Mono), Eosinophils (Eosin) and Basophils (Baso)). The red series andthe white cells were assayed using a calibrated Advia 2120 (Abbott)analyser.

In addition to cell counts we also monitored a range of blood chemistrycomponents: Fibrinogen, Serum Amyloid A, Creatine Kinase (CK), AspartateAmino Transferase (AST), Urea, Creatine (Creat), Total Protein (TotP),Glutamate Dehydrogenase (GLDH), Gamma-Glutamyl Transaminase (GGT),Alkaline Phosphatase (ALP), Lactose Dehydrogenase (LDH), Globulin (Glob)and Albumin (ALB). The fibrinogen was measured using a calibrated ACLElite analyser from Instrumentation Laboratory. SAA was measured using acalibrated Konelab 20 instrument from Thermo Scientific with the ‘Eiken’Serum Amyloid A test reagents supplied by Mast Diagnostic. The Eikenassay is a human immunoturbido metric method which has been previouslyvalidated in horses [20]. According to the manufacturer the range of thetest is 5-500 ug/ml with a coefficient of variation for less than 10%and an accuracy of 85-115% when a known concentration is measured. Themeasurement of 57 samples reported a correlation coefficient (r) asr=0.981 and the regression line as y=0.971x+2 [21].

All tests were performed by the suitably qualified in-house labtechnician. To minimise the impact of circadian fluctuations and toallow for horses to return to the resting state, blood was drawn between2 pm and 3 pm according to in-house procedures and veterinaryrecommendation by the in-house vet. The blood was drawn into blood tubesappropriate for the parameters to be tested. The results for each of theparameters under analysis in this study for each of the 223 horses werecompiled and analysed using Microsoft excel.

3. Results

The three primary measures obtainable from blood that we were mostinterested in were the classical total white cell count and two proteinsassociated with the inflammatory response, fibrinogen and SAA. We beganby asking whether, across the entire range of observed values, there wasa general tendency for high and low values in one measure to beassociated with high and low values in another. Since several of thetrait value distributions were strongly non-normal we usednon-parametric rank correlation tests rather than a standard Pearsoncorrelation.

Rank correlations between our three primary measures and all othertraits are presented in Table 1. Among the three primary measures, thetwo indicators of inflammation correlate positively and highlysignificantly with each other, but there is no association betweeneither of these and WBC. As might be expected, WBC counts are positivelycorrelated with many of the other sub-classes of blood cell counts,particularly neutrophils, lymphocytes and red blood cells. Among theblood chemistry measures, WBC is associated with GLDH and ALP, while theinflammation proteins both correlate with total protein and globulin,but also exhibit weak correlations with several others. Red blood cellsare interesting, since they correlate positively with WBC and SAA butnegatively with fibrinogen.

From the point of view of diagnosing imminent health issues, weakcorrelations between two or more measures across all horses may or maynot be biologically relevant. For example, mild dehydration might resultin transiently higher protein concentrations across many/most molecules,and this could drive correlations even across a sample of equallyhealthy animals. More clinically relevant, therefore is the tendency formeasures to show concordance when levels have risen outside what mightbe considered the ‘normal’ range of values. We first explored publishedtables giving the ‘normal ranges’ for different classes of horse (e.g.‘thoroughbreds’ or ‘2 year olds in training’) but several traits inthese systems were in contradiction with one another and routinelyyielded values outside the expected ranges depending on which referencemethod was applied. Consequently, we turned to a more unbiased approach.We arbitrarily assumed that the highest 15% of observed values for eachparameter were ‘elevated’ and used simple chi-squared tests to askwhether these elevated values at our three focal variables tended to beassociated with elevated values in each of the other traits. 15% waschosen as a balance between a lower fraction that would have too littlestatistical power and a higher fraction that might be deemedunrealistic. This method therefore bypasses the need to predefine‘normal’ and ‘abnormal’.

The chi-squared tests for concordance of high values are summarised inTable 2. With (overly) stringent full Bonferroni correction forconducting 63 tests, four tests are significant experiment-wide: Fib vSAA (X²=43.7, 1df, P=1.4×10⁻¹¹), WBC v Neutrophils (X²=27.9, 1df,P=1.3×10⁻⁷), WBC v Lymph (X²=13, 1df, P=3.2×10⁻⁴) and WBC v ALP(X²=11.4, 1df, P=7.5×10⁻⁴). In addition, a number of other combinationsyield significance at P=0.05 uncorrected, noticeably Total Protein,Neutrophils and Globulin, which all show associations with all three ofour primary measures. It is reassuring that the strongest association,using both statistical models, by some way is the one between fibrinogenand SAA, the two measures of the inflammatory response. In all cases theassociations are positive, in that the highest values for one traitoccur disproportionately frequently with high values at another trait.

TABLE 1 Correlation between values in diverse blood assays in 224thoroughbred racehorses. Two proteins associated with the inflammatoryresponse, Serum Amyloid A and Fibrinogen, and total White Cell Count arecompared against each other and against 20 other cell count/proteinassays. In each case a non-parametric Spearman rank correlation isperformed. Values presented are the resulting P-values. Valuessignificant at P < 0.05 are indicated with one asterisk, thosesignificant experiment-wide are indicated with two asterisks. Assayabbreviations are found in methods. Fibrinogen SAA WBC SAA 1.1 × 10−⁰⁹**WBC 0.95 0.21 Neut 0.92 0.41 3.8 × 10⁻²⁸** Lymph 0.005* 0.01* 3.0 ×10⁻⁰⁸** Mono 0.08 0.09 1.3 × 10⁻⁰⁴** Eosin 0.76 0.49 0.28 Plt 0.03* 0.390.01* Baso 0.02* 0.03* 0.41 RBC 0.02* 2.69 × 10⁻⁰³* 7.8 × 10⁻⁰⁷** Hgb0.45 0.03* 3.1 × 10⁻⁰⁵** Hct 0.46 0.03* 1.1 × 10⁻⁰⁴** TotP 3.9 × 10⁻⁰⁵**0.02* 0.10 Creat 0.31 0.17 0.38 Urea 0.02* 0.45 0.26 GGT 0.09 0.82 0.27AST 0.33 0.02* 0.03* CK 0.009* 0.03* 0.13 LDH 0.04* 0.04* 0.09 GLDH 0.060.42 7.5 × 10⁻⁰⁵** ALP 0.10 0.09 1.1 × 10⁻¹²** ALB 0.50 0.18 0.41 Glob2.2 × 10⁻⁰⁷**  2.5 × 10−03* 0.18

TABLE 2 Concordance of occurrence of extreme values among diverse bloodassays. Two proteins associated with the inflammatory response, SerumAmyloid A and Fibrinogen, and total White Cell Count are comparedagainst each other and against 20 other cell count/protein assays. Ineach case a simple 2X2 test of homogeneity is conducted to test for anassociation between the top 15% of values observed. Values presented areinterpreted with one degree of freedom. Values significant at P < 0.05are indicated with one asterisk, those significant experiment-wide areindicated with two asterisks. Abbreviations of assays are found inmethods. Fibrinogen SAA WBC SAA 45.7** WBC 1.1 4.2* Neut 6.1* 7.2*27.9** Lymph 2.9 1.8 13.0** Mono 2.1 1.4 6.2* Eosin 0.0 0.0 0.0 Plat 0.00.0 0.5 Baso 4.4* 1.3 0.0 RBC 2.9 0.7 0.9 Hgb 3.4 1.0 0.5 Hct 1.7 1.00.0 TotP 3.9* 5.6* 8.7* Creat 0.4 0.9 0.8 Urea 5.0* 0.0 0.0 GGT 0.4 0.10.1 AST 0.5 2.5 0.2 CK 0.0 0.1 0.0 LDH 2.3 2.1 0.5 GLDH 0.2 6.5* 1.5 ALP2.3 1.9 11.4** ALB 1.1 0.9 1.2 Glob 3.9* 9.4* 8.7*4. Discussion

We explored the relationship between a number of standard bloodparameters in a sample of thoroughbred racehorses in training. Our datareveal that while the most commonly used indicator of health, totalwhite cell count, correlates broadly with both individual cell sub-typecounts and several elements of blood chemistry, there is relatively pooragreement between horses with the highest white cell counts and thehighest values in other measures such as the inflammatory markers SAAand fibrinogen. In contrast, two components of the inflammatoryresponse, SAA and fibrinogen, correlate relatively weakly with WBC andblood chemistry but show excellent agreement with one another when itcomes to high values. Furthermore, by application of two separatestatistical models of analysis, similar trends can be observeddemonstrating that this study group was indeed a random samplepopulation of thoroughbred racehorses and may not have been overlyinfluenced by particularly ‘extreme’ individuals.

Blood chemistry and white cell counts are both used routinely asindicators of health however readings in healthy horses are far fromconstant and vary with levels of hydration and other factors. For thisreason, measurements are generally conducted in as standardised a way aspossible, at the same time of day and the same time relative to feedingand exercise. Nonetheless, variation still seems likely due to factorssuch as individual-specific patterns in urination, environmentaltemperature and anxiety, and this appears to be reflected in the waymost of the white cell counts and blood chemistry measures exhibit somedegree of cross-correlation.

To understand which part of the range of observed values of a giventrait are associated with ill-health as opposed to natural daily andhourly variation in homeostasis would involve tracking the fate ofhorses that were trained at a constant level until clinical symptomsdeveloped. However, such an experiment is largely precluded by the needto act pre-emptively so as to maximise horse welfare. Instead,therefore, we focused entirely on correlations between the various bloodanalytes in general (Table 1), comparing these with the level ofconcordance seen between high value readings for the same measurements(Table 2). In this way we can see the extent to which differentmeasurements co-vary across their entire range, a pattern that wouldsuggest correlation with some other factor such as diurnal variation inhydration, as opposed to a specific tendency for high values at onemeasure to be associated with high values at another, a pattern thattends to identify an unusual subset of horses. We presume that suchsubsets represent horses with, in this case, an on-going inflammatoryresponse.

Our argument is that, from experience, a small but unknown subset of ournumber of horses in training are likely to have incipient health issues.If these horses can be detected, they should be contributing unusuallyhigh trait values. Moreover, if two or more traits are useful asindicators, these should show good agreement in their highest values.When we interrogate our data in this way we find a reversal, with WBCshowing weaker correlations among the highest 15% of values comparedwith fibrinogen and SAA. By implication, fibrinogen and SAA showagreement in identifying a subset of horses with unusual readings, mostparsimoniously explained by these horses currently suffering some levelof injury or illness involving the inflammatory response. The apparentlack of specificity of WBC counts likely reflects the large diversity offactors that can affect them, many of which are not directly related tohealth.

Our results raise questions both about what WBC are detecting and whatthey are expected to detect as a pre-performance assay. Cell countsundoubtedly fluctuate in a biologically meaningful way, but there aretwo complications. First, the correlation between WBC and many of theblood chemistry measures suggests that the majority of the variation inour sample is due to normal variation in blood concentration rather thanspecific responses to a particular challenge. Second, the range ofstimuli capable of impacting WBC is wide, diverse and some may evendepress cell counts. Consequently, a single WBC is unlikely to tell usmuch about incipient problems. Better would be a monitoring programmebased on repeated measures so that sudden changes could be betteridentified, but even here the meaning of such changes may be difficult.

In comparison with WBC, fibrinogen and SAA appear to have considerablybetter discriminatory power, both largely agreeing with each other abouta subset of horses with clearly elevated readings. The implication isthat these horses may have an otherwise undetected health problem. Froma diagnostic perspective, this brings both positive and negativeaspects. The negative aspect is that SAA and fibrinogen will notidentify horses suffering from problems that are not currently causingan inflammatory response. The positive side is that these two bloodproteins, in contrast to WBC, appear to identify a relatively specificstate, that of horses exhibiting an inflammatory response.

5. Conclusions

We conclude that fibrinogen and SAA have excellent potential asbiomarkers and are likely to be more informative about conditionsrelevant to horses in training compared with the widely used WBC.

REFERENCES FOR EXAMPLE 1

-   [1] Parry-Billings M, Budget R, Koutedakis Y, Blomstrand E, Brooks    S, Williams C, et al. Plasma amino acid concentrations in the    overtraining syndrome: possible effects on the immune system. Med    Sci Sports Excer 1992; 24:1353-1358.-   [2] Rickets S W. Hematologic and Biochemical abnormalities in    athletic horses. In: Hinchcliff K W, Keneps A J, Geor R J, editors.    Equine Sports Medicine and Surgery, Philadelphia: W. B Saunders;    2004, p. 952.-   [3] Welles E G. Interpretation of Equine Leukocyte Responses. In:    Weiss D J, Wardrop K J. Schalm's Veterinary Hematology, 6.ed, Iowa:    Wiley-Blackwell; 2010, p. 317-   [4] Couetil L L, Hoffman A M, Hodgson J, Buechner-Maxwell V, Viel L,    Wood J L N, et al. Inflammatory Airway Disease of Horses. J Vet    Intern 2007; 21:356-361.-   [5] Grondin T M, Dewitt S F, Normal hematology of the horse and    donkey. In: Weiss D K, Wardrop K J. Schalm's veterinary hematology,    6.ed, Iowa: Wiley-Blackwell, 2010; p. 821-828.-   [6] Hultén C, Sandgren B, Skioldebrand E, Klingeborn B, Marhaug G,    Forsberg M. The acute phase protein serum amyloid A (SAA) as an    inflammatory marker in equine influenza virus infection. Acta Vet    Scand 1999; 40:323-333.-   [7] Hulten C, Gronlund U, Hirvonen J, Tulamo R M, Suominen M M,    Marhaug G, et al. Dynamics in serum of the inflammatory markers    serum amyloid A (SAA), haptoglobin, fibrinogen and alpha2-globulins    during induced non-infectious arthritis in the horse. Equine Vet J    2002; 34: 699-704.-   [8] Pepys M B, Baltz M L, Tennent G A. Serum amyloid A (SAA) in    horses: objective measurement of the acute phase response. Equine    Vet J 1989; 21:106-109.-   [9] Jacobsen S, Nielsen J V, Kjelgaard Hansen M, Toelboell T,    Fjeldborg J, Halling Thomsen M, et al. Acute phase response to    surgery of varying intensity in horses: a preliminary study. Vet    Surg 2009; 38:762-769.-   [10] Pusterla N J, Watson J L, Wilson W D. Diagnostic approach to    infectious respiratory disorders. Clin Tech Eq Pract 2006;    5:174-186.-   [11] Allen B V, Kold S E. Fibrinogen response to surgical tissue    trauma in the horse, Equine Vet J 1988; 20: 441-443.-   [12] Burrows G E. Dose-response of ponies to parenteral Escherichia    coli endotoxin. Can J Comp Med 1981; 45:207-210.-   [13] Crisman M V, Scarratt W K, Zimmerman K L. Blood Proteins and    Inflammation in the horse. Vet Clin Equine Practice 2008;    24:285-297.-   [14] Heidman P, Madigan J E, Watson J L. Rhodococcus equi Pneumonia:    Clinical Findings, Diagnosis, Treatment and Prevention. Clin Tech Eq    Pract 2006; 5:203-210.-   [15] Takizawa Y, Hobo S J. Usefulness of plasma fibrinogen    concentration measurement in diagnosis of respiratory disorders in    thoroughbred horses. Equine Sci 2006; 2:22-37.-   [16] Satue K, Calvo A, Gardon J. Factors Influencing Serum Amyloid    Type A (Saa) Concentrations in Horses. Open Journal of Veterinary    Medicine 2013 3:58-66.-   [17] Tape C, Kisilevsky R. Apolipoprotein A-I and apolipoprotein SAA    half-lives during acute inflammation and amyloidogenesis. Biochem    Biophys Acta 1990; 1043:295-300.-   [18] Vandenplas M L, Moore J N, Barton M H, Roussel A J, Cohen N D.    Concentrations of serum amyloid A and lipopolysaccharide-binding    protein in horses with colic. Am J Vet Res 2005; 66:1509-1516.-   [19] Jacobsen S, Thomsen H, Nanni S. Concentrations of serum amyloid    A in serum and synovial fluid from healthy horses and horses with    joint disease, Am J Vet Research 2006; 67:1738-1742.-   [20] Jacobsen S, Kjelgaard-Hansen M, Petersen H, Jensen A L.    Evaluation of a commercially available human serum amyloid A (SAA)    turbidometric immunoassay for determination of equine SAA    concentrations. Vet J 2006; 172(2):315-319.-   [21] Mast Group (No publication date) Eiken Serum Amyloid A    (SAA)[Online] Merseyside, Mast Group LtdAvailable:    http://www.mastgrp.com/Eiken/InfoSheet/SAA %20reagents.pdf [Accessed    18 Sep. 2013].

Example 2

Introduction and Methods

Clinical symptoms in horses may only appear when over-stressing hasalready occurred, and there is an unmet need to provide methods todetermine imminent problems at sub-clinical stages.

The SAA levels of a group of thoroughbred horses bred for flat racingwere recorded over a three month period (April to June and n=61) as partof a routine biochemical panel for pre-performance testing. Of the 61horses tested 25 ran during the testing period. The horses were managedin the same way under the same training schedule. SAA levels weredetermined using a two-step lateral flow immunoassay and a lateral flowreader (LFR101) by the suitably qualified in-house laboratorytechnician. The names of the horses and the events in which theyparticipated were recorded but not reported. The data was observed andthree relevant ranges for horses in training became apparent. Horseswith a SAA concentration below 7.5 ug/ml are clinically well, free fromsubclinical infection and with the exception of those conditions whichdo not invoke a SAA response, are fit and healthy horses. Horses thatran with SAA levels of 15 μg/ml and over performed below expectation,particularly those with SAA levels in excess of 30 μg/ml. Horses with aSAA of greater than 200 μg/ml are clinically unwell with visiblesymptoms. The SAA levels of 16 horses in the study group was tested morethan once to monitor recovery and/or to further impaired performance.

Results and Discussion

In the group of horses that tested with a SAA concentration greater than200 μg/ml (n=5) (Table 1), 3 of the group had infections confirmed byeither clinical examination or further by diagnostic investigation. Onerecord for a recent inoculation of the equine herpes virus is reported.One horse of the group ran during the study and did not perform toexpectation. The horse was assumed to have virus and subsequent testingat a later date identified mucus and neutrophils in a tracheal wash(Table 2).

TABLE 1 Levels above 200 μg/ml - Elevated SAA post vaccination andclinical confirmation of infection in 3 of the 4 horses who were notvaccinated. Subsequent testing of the remaining horse confirmed thepresence of bacterial or viral infection. SAA Comments on μg/mlInfection Performance Trainers Comments 641.7 n/a n/a Equine herpesvirus vaccination 539.6 Confirmed by n/a presence of bacteria in lungwash 331.7 Confirmed by n/a Infected leg wound clinical examination ofwound 243.4 Confirmed by n/a Cracked heels tracheal wash 234.72 Notconfirmed Performed lower Potential virus, visibly than expectations outof form

TABLE 2 A horse with an elevated SAA >200 μg/ml was monitored post-racefollowing a poor performance. Neutrophils were subsequently identifiedin a tracheal wash. SAA had returned to normal levels after antibiotictreatment. SAA Date μg/ml Trainer Comments 18 Apr. 2013 234.72 Performedbelow expectation - suspected virus 25 Apr. 2013 63.46 1 May 2013 128.47Mucus and neutrophils in tracheal wash 7 May 2013 1.38 Post antibiotictreatment

SAA concentrations between 30 μg/ml and 200 μg/ml were recorded for 15(Table 3) horses in the study group. 6 of the 16 ran with an elevatedSAA and all performed lower than the expected standard according tocomments recorded at the time of testing. Post-race testing revealedmucus and blood in the tracheal wash of one of the 6 runners whileanother with a known elevated SAA concentration had been diagnosed witha bacterial lung infection 10 days prior to the race. In this instanceSAA was monitored and levels had decreased but remained elevated on theday of racing and performance was recorded as below expectations.

SAA levels were monitored over the course of 9 days for one horse in thegroup of 6 under-performing horses (Table 4). Oμg/ml was recorded on theday of racing however SAA had increased to 32.46 μg/ml and 34.01 μg/mlon day 2 and 3 respectively with no clinical symptoms were detected. SAAlevels had returned to Oμg/ml by day 9 of testing. A knee injury wassustained by one of the runners during the race and in addition asuspected viral infection was recorded for the same horse. SAA levels,post-performance were measured at 175 μg/ml which supports thelikelihood of a pre-existing of viral infection. SAA was the soleindicator of a subclinical challenge for 3 of the 6 underperformers.

Of the horses who did not run within this range no clinical symptomswere recorded at the time of testing for two of the group however theremaining 8 horses, 4 were being monitored, 1 had an abnormal trachealwash without confirmation of infection and 2 had no clinical symptoms.Comments were not recorded for one horse with an elevated SAA.

TABLE 3 SAA concentrations above 30 μg/ml but below 200 μg/ml (wheresymptoms are visible) indicate the presence of an underlying issue. SAAPerformance μg/ml Infection Comments Trainer Comments 196.92 No clinicalsymptoms 189.17 No clinical symptoms 175.39 Performed Knee injurysustained below during race. Suspected expectation virus. *128.47Confirmed by the presence of mucus and neutrophils in tracheal wash*119.42 Performed No clinical symptoms below expectation *108.23Confirmed Post antibiotic treatment bacterial infection in lungs *97.90Confirmed by bacteria in tracheal wash 93.59 Confirmed by bacteria intracheal wash 79.82 Performed No clinical symptoms below expectation65.18 Confirmed by white blood cells in tracheal wash *65.18 Confirmedby Performed bacterial infection below in lungs expectation *63.46Confirmed by the SAA is decreasing presence of mucus and neutrophils intracheal wash 55.71 *52.26 Confirmed by Post antibiotic treatmentbacteria in tracheal wash 52.26 Confirmed by Performed Blood in trachealwash mucus in tracheal below wash expectation 46.41 46.24 Performed Noclinical symptoms below A very consistent expectation flat horse *34.01*32.46 31.60 Not confirmed Abnormal tracheal wash Horses which appeartwice within the table are marked with as asterisk*.

TABLE 4 A horse was monitored was 9 days following a poor performance,SAA levels had returned to normal by day 9 of testing. There were noclinical symptoms. SAA Date μg/ml Trainers Comments 21 May 2013 0Performed below expectation 22 May 2013 32.46 — 23 May 2013 34.01 — 30May 2013 0 —

Seven horses were recorded with an SAA concentration between 15 μg/mland 30 μg/ml (Table 5). 3 of the 7 raced during the duration of thestudy and 2 performed below expectations. There were no visible symptomsof illness observed in the runners and further investigation revealedonly an elevated AST concentration for one of the pair. Of the remaininghorses that did not run, all were under investigation for previous poorperformance and SAA levels were elevating or decreasing when recorded.

TABLE 5 SAA concentrations between 15 ug/ml and 30 ug/ml. SAAPerformance μg/ml Infection Comments Trainer Comments *29.88 Fibrinogenelevated 28.16 Confirmed by AST elevated presence of bacteria intracheal wash 28.16 Performed below No clinical symptoms expectation*25.92 Performed as Blood in tracheal wash expected 24.71 Not confirmedAbnormal tracheal wash Antibiotic treatment *19.55 18.68 Performed belowAST elevated expectation *17.82 15.24

3 SAA concentrations were recorded for horses between 7.5 μg/ml and 15μg/ml. One horse of the group ran performing as expected. The tworemaining horses did not run and were being monitored following aninjury and an abnormal tracheal wash (Table 6). Horses within this rangemay be in the initial stages of an SAA elevation or they may berecovering from an infection where concentrations have droppedsignificantly. For this reason it is difficult to realise the potentialof SAA when used within this range. A more reliable range begins at 15μg/ml above which poor performance is likely when considering this setof data.

TABLE 6 SAA concentrations between 7.5 μg/ml and 15 μg/ml SAAPerformance Date μg/ml Infection Comments Trainers Comments 09.05 14.38Not confirmed Blood in tracheal wash 18.05 11.80 Knee injured 19.04 7.75Performed as expected

69 SAA levels were recorded below 7.5 ug/ml for 55 horses. SAA levelswere being monitored for 14 of the group. 13 ran during the study periodand with the exception of one horse that later measured with an elevatedSAA, all performed as expected. Outside of those horses being monitoredonly one confirmed infection was reported.

TABLE 7 SAA concentrations below 7.5 μg/ml SAA Performance μg/mlInfection Comments Trainer Comments 7.49 Performed as expected 7.49 4.05Performed as expected 3.19 Chronic lung problems 3.19 Performed asexpected 1.81 Performed as expected 1.38 Post antibiotics treatment 0.60Confirmed by presence of mucus and neutrophils in tracheal wash 0.43 0Abnormal profile 0 Performed as expected 0 Out of form 0 Performed asexpected 0 0 Performed below expectation Blood in tracheal wash 0 0Fibrinogen elevated 0 Fibrinogen elevated 0 0 Injured knee 0 Performedas expected 0 0 0 0 0 0 Lung allergy 0 0 0 0 Post antibiotic treatment 0Blood in tracheal wash 0 Lung allergy 0 Lung allergy 0 Performed asexpected 0 Performed as expected Abnormal tracheal wash 0 Lung allergy 0Blood in tracheal wash 0 0 0 Performed as 0 Infection expected Blood andmucus in tracheal wash confirmed by mucus in tracheal wash 0 0 Performedas expected Performed as expected 0 0 0 Post antibiotic treatment 0Confirmed by neutrophils in tracheal wash 0 Elevated AST

The SAA levels of a number of horses were recorded on more than oneoccasion during the study in order to monitor recovery. Those horses forwhich four or more data points were recorded are reported here (Table8). The data indicates that SAA levels resolve before the traditionalWBC profile returns to normal. In addition SAA can be used to determinethe efficacy of a treatment by monitoring the response of the proteinpost administration.

TABLE 8 14 horses were monitored to access SAA as a tool for monitoringrecovery during the study. SAA Date Name μg/ml Trainer Comments 24.0428.16 Mucus and bacteria in tracheal wash 26.04 0 WBC blood profile isstill abnormal 30.04 0 02.05 0 21.05 0 Performed below expectation 22.0532.46 23.05 34.01 30.05 0 SAA normal 26.04 93.59 Abnormal tracheal wash02.05 24.71 On antibiotics 14.05 0 Post antibiotic treatment 24.04343.29 Bacteria in tracheal wash, cracked heels 30.04 0 09.05 0 11.06501.72 Bacterial infection in lungs 11.06 108.23 Post antibiotictreatment 21.06 65.18 Performed below expectation. 09.05 14.38 Increasedblood in tracheal wash 14.05 0 SAA normal 22.05 52.26 Performed belowexpectation 30.05 0 SAA normal 04.06 394.95 Infected leg wound 11.06 0SAA normal

The vast majority of horses are physiologically healthy and this isreflected in the data. As SAA concentration exceeds 7.5 μg/ml theperformance of the horses in the study decreased with few exceptionsmaking SAA a very relevant biomarker for horses in training. The datafor the performance of horses between 7.5 μg/ml and 15 μg/ml is ratherinconclusive and performance is hard to predict. Above 15 μg/ml adecline in performance persists however other indicators of a reducedphysiological health status do not always accompany the measurement. Aparticular decline in performance is noted above 30 μg/ml and elevatedSAA is more often accompanied by other indications of infection mostnotably a tracheal wash elevated neutrophil count. As SAA concentrationsfurther increase clinically visible symptoms of illness become apparentand above 200 μg/ml, all records were accompanied by visible illnessand/or additional abnormal test results.

Three relevant ranges have emerged, <7.5 mg/ml, 15 ug/ml and >200 ug/ml.Since a decline in performance is most notable above 15 μg/ml, a usefultool for determining SAA does not indicate the presence of the proteinuntil levels have reached or exceeded this value to facilitate ease ofinterpretation. An increase in the test signal should correspond to anincrease in SAA levels and be present in the form of a single band. Sucha testing format makes the user immediately aware that SAA in present ina concentration which may require attention when a signal appears. Theabsence of a signal indicates to the user that the horse is in a healthystate. The SAA concentration can then be semi-quantitatively determinedwith the use of a reference card demonstrating levels of intensity towhich the test signal can be compared or a quantitative reading can bedetermined with the use of an electronic reader.

Example 3

A case study was conducted to assess the efficacy of SAA as a tool tomonitor and manage the recovery of horses. A previous study hadindicated that SAA concentrations above 200 ug/ml are accompanied byclinical symptoms and therefore SAA may be used monitor recovery andresponse to treatment; however the range above 200 μg/ml was not fullyinvestigated due to the limitation of elevated samples.

For this reason an additional case study was performed to specificallyevaluate SAA as a tool for monitoring recovery. Clinical comments wereprovided by a veterinarian at the time of testing. The name of the horsewas recorded but is not reported.

The SAA levels of a horse that presented with travel sickness(pleuropneumonia) were measured daily for 13 days following a 6689kilometer transportation (Table 1, FIG. 11). Clinical examination, SAAtesting and scanning were carried out by an ambulatory veterinarian. Thehorse was treated with Ceftiofur (antibiotic), Marbofloxacin(antibiotic), Flunixin (anti-inflammatory) Metronidazole (antibiotic)and Gastrogard (treatment/prevention of equine ulcers). The rapidincrease on day 7 corresponds with a neck injury from treatmentadministration.

TABLE 1 SAA measurements and clinical assessment of a horse with travelsickness. SAA Date μg/ml Veterinarian Comments 21.06 379.5 Temp 102,thin, dull. Fluid on right hand side chest & consolidated lung. 22.063493 Improved condition, temp 101. Lung consolidation. 23.06 3285 Tempnormal 24.06 3384 Temp normal, consolidation resolving 25.06 3112 Tempnormal, consolidation resolving 26.06 2640 Temp normal, consolidationresolving, reduced flunixin 27.06 3960 Temp normal, consolidationresolving, reduced flunixin 28.06 2755 Gas pocket in neck, changedceftiofur to cefquinome 29.06 838 Scan much improved 30.06 475 Tempconsistent 99.8 01.07 199 Temp consistent 99.8 02.07 101 Temp consistent99.4, stop flunixin 03.07 39 Scan improved, small comet tails, stopmarbocylDiscussion

The data from the case study indicate that SAA can be used toefficiently monitor the recovery of the horse. The study particularlydemonstrates the use of SAA to determine the biochemical efficiency ofthe course of treatment and SAA elevations and decreases were inagreement with clinical examination.

Assay Development

A two-step lateral flow immunoassay was developed for horses after theobservation of data which supported the use of SAA as a pre-performancetest and as a health management tool for monitoring recovery and/orresponse to treatment. During the course of the study it became apparentthat fit and healthy horses were reported with SAA concentrations lessthan 7.5 μg/ml while impaired performance largely corresponded withlevels above 15 μg/ml. Levels in excess of 200 ug/ml were accompanied byclinical symptoms, the resolution or exacerbation of which was reflectedby the level of SAA.

The lateral flow assay was developed in the sandwich format and consistsof a nitrocellulose membrane upon which an anti-SAA antibody and acontrol antibody have been immobilised. The membrane is assembledtogether on a backing material with a glass fibre conjugate pad, acellulose sample pad and a cellulose absorbent pad. The conjugate padcontains the anti-SAA-colloidal gold complex which is required for thedetection of SAA. The sample pad contains additional reagents whichincrease the stability and performance of the assay. The materials areassembled together into a plastic housing which consists of a samplewell and a viewing window. Prior to sample application, the sample isdiluted 1/800 in a running buffer (5 ul in 4 ml). The sample is appliedto the sample pad via the sample well. The reagents within the sampleand conjugate pad become mobile and move through the membrane to testline where a signal is raised if SAA is present at or above 15 μg/ml inthe sample. The intensity of the test line visibly increases as theconcentration of SAA increases up to a visible maximum 1000 μg/ml wherethe line becomes saturated to the eye. The range can be further extendedup to 3000 μg/ml using an electronic reader. A semi-quantitative readingcan be determined by use of a reference card upon which representationsof the intensity of 15 μg/ml, 50 μg/ml, 200 μg/ml and 1000 μg/ml areavailable for comparison.

Example 4—SAA to Distinguish Infectious and Non-Infectious Disease orSyndromes

Introduction and Methods

A number of case studies were compiled from data generated through twoEquine Veterinary Hospitals. SAA levels were determined by the in-houselaboratory technicians and clinical comments were provided by BoardCertified Internal Medicine Veterinarians

The levels of SAA were determined in horses diagnosed with infectiousand non-infectious diseases and was observed to respond most rapidly anddramatically to bacterial and viral infections, while allergies, EIPHand other non-infectious inflammatory conditions showed little or noresponse. SAA levels were also observed to elevate during colic andpost-colic surgery indicating SAA as a potent marker of infection andnot a marker general inflammation.

Results and Discussion

Infectious

SAA Peak No. of Range Common Case Diagnosis Detected Symptoms ObservedStudies Bacterial Lung 45-1028 Bacteria in trach wash 6 InfectionUnidentified Viral 16-1109 Fever, filled legs 10 Infection Rhodococcusequi 709-4936  Mucus, Cough 7 Rotavirus 1416-2763  Diarrhea, loss 2 ofappetite Post-Colic Surgery 100-1000+ Discomfort, 3 Infection SwellingPost-Gelding 709-4453  Swelling 7 Infection Abscess 14-4868 Swelling,Heat. 5 Cellulitis 4931 Heat, Pain. 2 Encephalitis 2838 Fever 1Osteomyelitis 329-905  Swelling, Lameness 3 Pneumonia 141-5000+ Cough,Fever 5 Peritonitis  5000+ Abdominal pain 1Non-Infectious

SAA Peak No. of Range Common Case Diagnosis Detected Symptoms StudiesExercise Induced 0-45  Blood in trach wash 3 Pulmonary HemorrhageAllergy 0 Snorting, coughing. 1 Blood in trach wash. Colic 100-1000+Abdominal pain 3 Inflammatory Bowel 10 Abdominal pain 1 Disease AirwayInflammation 2.2 Cough, Mucus 1 Edema 0 Swelling 1 Exertional 0Lameness, Cramping. 52 Rhabdomyolysis Heaves 0 Cough, Mucus 1

The case studies compiled in Table 1 demonstrates the potential forserum amyloid a to be used as a method for differentiating betweeninfectious and non-infectious conditions.

Levels of SAA detected for EIPH are considered to be from early stagesof a secondary bacterial infection.

The benefits of such a method extend to diagnostic procedures where aninfection can be confirmed before further investigation as well asallowing for the prompt initiation of a suitable treatment regime forsick horses based on whether they are being treated for an infectiousdisease such as those associated with micro-organisms or non infectiousillness such as those associated with the environment or lifestyle orgenetic factors. Furthermore SAA has been seen to elevate to a largerextend when the horse is challenged with a bacterial infection comparedto a viral infection which creates scope for SAA to be used not only asa marker of differentiation between infectious and non infectiousdisease but also as a method of differentiating between the organismresponsible which has implications for the type of therapy administerede.g. viral infections will not respond to antibiotic therapy.

Example 5—SAA as a Screening Tool in Newborn Foals

Introduction

Screening for SAA in newborn foals, under 10 hours old, has been shownto be an excellent risk-reduction method and can clearly identify foalssusceptible to liver failure, diarrhea and other infectious conditions.

SAA (Serum Amyloid A) was adapted as part of a health screening test fornewborn foals. A white blood cell count (WBC) was also conducted as partof the screening process. As WBC naturally fluctuate and can go down andup when challenged, SAA as a point of care screening tool is a morereliable indicator of a newborns changing health status.

Methods

Testing was conducted by the suitably qualified in-house laboratorytechnician and clinical comments were provided by a licensedVeterinarian within 24 hours of birth. Data was collected from 22newborns in total and analysed to determine the potential for SAA as ascreening tool for compromised newborns.

Results and Discussion

22 newborn foals were screened of which 15 tested SAA negative and 7tested SAA positive (Table 1). WBC data for the newborns ranged from7.1-17.9×10³/μl.

TABLE 1 Blood results and clinical notes from 22 newborns SAA Case No.(μg/ml) Clinical Notes 1 16.5 Healthy newborn, developed R. Equi onemonth later. 2 0 Healthy newborn 3 7.5 Healthy newborn, went on todevelop multiple joint and bone infections. 4 0 Healthy newborn 5 0Healthy newborn 6 0 Healthy newborn 7 0 Healthy newborn 9 0 Healthynewborn 10 0 Healthy newborn 11 81.5 Healthy newborn 12 0 Healthynewborn 13 0 Healthy newborn 14 1464.5 Rotavirus diagnosed and sent tohospital 15 0 Healthy newborn 16 0 Healthy newborn 17 265 Elevated BUNand creatine 18 0 Healthy newborn 19 0 Healthy newborn 20 0 Healthynewborn 21 35.5 Healthy newborn, went on to develop diarrhea 22 167.5Weak

Of the 7 SAA positive newborns, 1 was diagnosed within 24 hours of birthwith a viral infection and transferred to hospital. Another displayedelevated BUN and creatine levels in addition to elevated SAA.

A third newborn was described as weak. Two newborns went on to develophealth problems a later date, one within the first month of birth andone within two weeks.

The newborn in Case 1 (Table 1) was diagnosed with Rhodococcus equi onemonth after the SAA determination. The newborn was determined to behealthy on examination and had a mildly elevated SAA level. Rhodococcusequi is a bacterial infection and one of the most common causes ofpneumonia in foals. Infected foals may remain lively and asymptomaticuntil late in the course of the disease. Infection has been recognizedas endemic on some farms and costs related to illness and mortality maybe high at these locations Rhodococcus equi is nearly ubiquitous in soiland while the infection is unlikely to have been contracted immediatelyafter birth, SAA can be used in as a screening test for earlyidentification of those newborns who may be at risk of developing theinfection.

The newborn in case 3 was assessed as a healthy newborn after birth andhad a low level of SAA. Within two weeks of initial SAA testing the foalhad developed multiple joint and bone infections during which SAA levelsrose in excess of 3000 μg/ml.

In addition to be being used as a screening tool to identify potentiallycompromised newborns SAA was also used in this instance to determine thepoint at which antibiotic treatment should be withdrawn by monitoringthe decrease in SAA levels.

SAA has been shown to detect the presence of viral infections includingrotavirus. Rotavirus is a highly contagious virus that affects foals andif left untreated can become life threatening due to severe dehydrationand malnutrition. Case 14 provides a second example of the potential forSAA to be used as an initial screening tool and then as a tool tomonitor the response of a foal to treatment. The newborn was diagnosedwith a rotavirus infection within 24 hours of birth and sent tohospital. SAA levels were then recorded until they fell within thenormal range.

Unlike adult horses, newborns may display levels of SAA elevation in thevery early hours of life due to liver activity unrelated to infection,its assumed that this may be related to the transfer of functionalityfrom the placenta to the newborn liver as can been observed in case 17below, where markers of poor liver function are elevated. Neverthelessit can be seen that SAA is a useful marker of foals that may develophealth issues within the first 48 hours after birth, which is when foalsare at highest risk of fatality.

Example 6—Test Fluid Collection System

Some key features of the system are shown in FIG. 14, and are explainedin more detail below:

Multi-Purpose Sample Collection Tip

The sample collection tip has been designed such that different methodscan be used to collect a sample. Firstly, the dimensions of the tipallow for the attachment of a luer end needle so that blood can becollected straight from the vein. Alternatively, a lancet can be used toproduce a drop of blood from the patient and the BCS used to pick up ametered volume from the drop using capillary action. Similarly, the BCScan pick up blood from the end of a syringe or from a container.

Collection Port

The BCS collection port can be considered to be the most important partof the device. The collection port comprises of two closely alignedsurfaces. The space between the two surfaces determines the volume ofsample to be collected. Due to the design of the port and nature of thehydrophilic-treated surfaces, sample collection occurs by capillaryaction and is quick and accurate. This removes the requirement foraspirating sample with a pipette, which removes the risk of error byusers who would not be familiar with using pipettes.

Dispensing Channel and Nozzle

The BCS is designed to fit into the neck of a bottle, with thecollection port and the dispensing channel located inside the bottle.The BCS/bottle can then be inverted for mixing. Reduction in volume ofthe bottle then forces the diluted solution into the dispensing channeland through the dispensing nozzle.

Example 7—Monitoring of Exertional Rhabdomyolysis

Exertional Rhabdomyolysis, also known as Tying up, is a conditioninduced by exercise, characterized by stiffness, hardened muscles in thehind quarters and reluctance to move.

The detection of elevated CK and AST levels in horses can be used todiagnose tying up, but the condition can be easily identified byphysical examination. The benefit of testing for CK and AST when a horseties up is the ability to monitor disease progression. If interpretedcorrectly, CK and AST levels can tell you if the horse is just beginningto tie up, whether it is responding well to an intervention or if thehorse is recovering.

Methods

A study conducted over a six-month period (May-November '13) testedapprox. 200 Thoroughbred horses bred for flat racing. During monthlyroutine blood testing, each horse had a complete blood cell count andbiochemistry panel conducted. Creatine kinase (CK) and aspartateaminotransferase (AST) were part of the biochemistry assessment. As wellas scheduled blood testing, additional tests were conducted to monitor ahorses CK and AST levels when tying up was observed.

Results

Over a period of 6 months, 52 out of 200 horses tied up, accounting for26% of the population. Of the 52 horses, 14 experienced recurrentepisodes of tying up, ranging from 2 to 5 cases in the six months. Thedata is tabulated below.

The incidence of recurrence is shown in FIG. 15.

The horses that experienced recurrent cases of tying up accounted for52.5% of the total 80 cases, indicating a requirement to monitor horsesprone to the condition.

Managing Exertional Rhabdomyolysis

Some horses are more prone to tying up than others, sometimesexperiencing episodes of tying up back-to-back, which can be frustratingto trainer, costing money and time. Over the duration of this study, 14horses were observed to have repeated episodes of tying up.

CK AST Clinical notes 6613 2279 Set fast 1236 1156 Set fast 1989 2380Set fast 4217 1444 Set fast 314 2441 Set fast 5998 1505 Tying up 57004451 Set fast 1948 1173 Set fast 1061 1027 Set fast 3780 1345 Set fast14291 1948 Set fast 4415 1042 Set fast 4781 1194 Set fast 2497 1590 Setfast 3875 3930 Set fast 12607 3457 Set fast 1198 2570 Tying up 1934 2740Tying up 528 2539 Set fast 3034 1043 Set fast 823 1288 Set fast 120351196 Set fast 11563 993 Set fast 9128 3984 Set fast 1132 3984 Set fast4191 3881 Set fast 577 3517 Set fast 1949 2386 Tying up 23166 2600 Setfast 8682 1720 Set fast 1133 2020 Set fast 6837 3090 Set fast 2329 1674Set fast 743 1462 Set fast 1015 923 Set fast 5719 1286 Set fast 1013 802Set fast 2806 1632 Set fast 6068 5190 Set fast 7947 5458 Set fast 81956940 Set fast 2086 2946 Set fast 5089 3743 Set fast 3010 3055 Set fast2307 2796 Tying up 4451 3181 Tying up 3755 1040 Set fast 720 1430 Setfast 1751 1996 Set fast 2159 1885 Set fast 0 1482 1962 Set fast 0 5501280 Tying up SAA CK AST Clinical notes 0 2638 688 Set fast 0 3692 920Set fast 0 1948 1139 Set fast 0 13391 5191 Set fast 0 16667 8549 Tyingup 0 6140 14479 Tying up 0 21541 20872 Tying up 0 1103 15327 Set fast 0346 11674 Set fast 0 263 11046 Set fast 0 1769 1068 Set fast 0 1832 909Set fastDevice for Managing Exertional Rhabdomyolysis

In one embodiment of the device (FIG. 17(a)) the device is a LFD and CKand AST are determined in sequence according to the positioning of thedetection reagents on the LFD.

In such a device sample is added to a single sample port and moves alonga channel where, for example, it first encounters the reagents requiredto detect and determine levels of AST and subsequently encounters thereagents necessary to detect and measure CK. In such a device CK and ASTmust both be measured, as the sample must come into contact with thereagents for the detection of each as it moves through the channel. CKand AST are clearly indicated (labelled) by markings on the device todifferentiate between the two (i.e “CK” is printed on the device at thetest line for CK and “AST” is printed on the device at the test line forAST).

In one embodiment of the device (FIG. 17(b)) the markings beside thetest line for CK appear as “CK” and the markings for AST appear as“SGOT” representing serum glutamic oxaloacetic transaminase by which ASTis also known. In this embodiment the CK marking is above the SGOTmarking.

In one embodiment of the device (FIG. 17(c)) CK and AST are measured ona LFD in parallel to each other. Such a device contains two distinctsample ports, which run in parallel to each other and which are not influid communication with each other. Sample is added in separate stepsto each sample port. The first of the two channels which make up thisembodiment contains only the reagents necessary for the detection of CK.The second of the two channels contains only the reagents necessary forthe detection of AST. Such a device allows for the analysis of CK/AST inparallel or individually. In the Figure the channel which detects andmeasures CK is visible on the left (front) side of the cartridge. Thechannel which detects and measures AST is visible on the right (front)side of the cartridge. The sample zones directly underneath the sampleports of the device may be treated with reagents which encourage theperformance of the test.

In one embodiment of the device FIG. 17(d)) CK and AST are determined bythe application of sample to a single sample port. The sample thenseparates and moves along two separate channels. The two channels arechemically treated in the same fashion as those described above wherebythe antibody reagents for the detection and measurement of CK arepresent only in the channel visible to the front left of the cartridgeand those for the detection of AST are present only in the channel iswhich is visible on the front right of the device. The sampleapplication zone of the device may be chemically treated in such a wayas to encourage the performance of the test.

It will be appreciated that the orientation (left) in the aboveembodiments is not essential.

Example 8—Presence of SAA in Healthy Horses

Introduction and Methods

A population study involving 105 thoroughbred horses was conducted inorder to understand the normal level of serum amyloid A in healthyhorses. The horses were a random mixture of males and females, a mixtureof grades, ranging in age from 2- to 5-year-old and had raced a maximumof five times each. All horses were managed in the same way withindividual boxes, photoperiod of 4:30 AM to 9 PM, a natural indoortemperature (18-C-20-C), and the same feeding and training schedules.Detailed veterinary analysis of each horse immediately after samplingwas beyond the scope of the present study. However, it was noted by theveterinarian that all horses were fit for work.

All blood analysis was performed by a suitably qualified in-houselaboratory technician. To minimize the impact of circadian fluctuationsblood was drawn between 2 PM and 3 PM according to in-house proceduresand veterinary recommendation by the in-house vet. The blood was drawninto blood tubes appropriate for the parameters to be tested.

SAA was measured using a calibrated Konelab 20 instrument from ThermoScientific and the “Eiken” test reagents supplied by Mast Diagnostic.According to the manufacturer the range of the test is 5-500 ug/ml witha coefficient of variation <10% and an accuracy of 85-115%.

The results for each of the parameters under analysis in this study foreach of the 105 horses were compiled and analyzed using Microsoft excel.

Results and Discussion

We considered the absolute presence or absence of serum amyloid A (SAA)in a population of 105 thoroughbred racehorses bred for racing. From 105subjects only 9 subjects were found to have any detectable level of SAA.

The lowest detected level was 5.4 μg/ml with 4 out of the 9 positive SAAresults under 25 μg/ml indicating that the sensitivity of the method wascapable of consistently determining concentrations in this range, and itwas considered that absolute negative results (zero) truly reflect thepractical absence of the protein.

The use of total white blood cell (WBC) counts, plasma fibrinogen (Fb)and SAA have been well reported for the diagnosis of inflammation inhorses. The normal ranges for WBC in thoroughbred racehorse has beenwell reported by Allen et. al. in 1984 who give various acceptableranges for fillies, colts and of different ages and stages of training.For the purposes of this study the normal ranges for WBC's wereconsidered to be 6.0-9.9×10⁹/L. Fb is an acute phase protein which isnow well established as a marker of inflammation in horses, initialreports for the use of fibrinogen as a marker of acute inflammation inhorses include van Wuijckhuise-Sjouke (1984) and Patterson et. al.(1988). Again various normal ranges have been reported and for thepurposes of this study 2.0-5.0 mg/ml is considered to be the normalrange. Pepys et. al. reported the first immunoassay for SAA in 1989 andconcluded that SAA was present only at “trace” levels in healthy horsesbut elevated rapidly following tissue injury (surgery) infection andinflammation. It is not clear whether the particular immunoassay used inthat work had the required sensitivity to establish if SAA was actuallyabsent.

Thus the present invention represents the first report that SAA isabsent in normal healthy horses.

96 out of 105 (91.5%) subjects gave absolute negative results for SAAindicating that the normal level of SAA is in fact none or zero. Foreach of the 9 positive results, the levels of WBC and Fb were alsoconsidered to report if an inflammatory response may have beenoccurring. It was also considered reasonable that in any population ofracehorses in training that up to 10% of the population may be sufferingfrom some kind of inflammatory process, albeit at the sub-clinical ormildly clinical stages.

5 of the subjects had highly elevated SAA levels (between 969-1868μg/ml) in each of these cases the Fb level was highly elevated (5.7mg/ml or higher).

Possibly most interesting is that the 2 subjects with SAA levels within5-20 μg/ml had corresponding WBC levels that were lower than the normalrange while the fibrinogen was either normal or mildly elevated. As SAAis understood to respond earlier than Fb, these two examples may reflectvery early stages of an inflammatory response, where WBC's becomedepleted in an immune response upstream of new WBC production, and,whereby Fb had not yet elevated. Subject 105 had an SAA level of 21μg/ml and corresponding elevated Fb level of 5.5 mg/ml. None of the 96SAA-negative horses had an elevated Fb measurement higher than 5.0mg/ml.

In summary, this example demonstrates that from a population of 105racehorses in training SAA is not at all present in normal healthyhorses and in horses where SAA is detected its likely that aninflammatory process had been triggered.

TABLE 1 WBC Fibrinogen SAA Horse (10⁹/L) (mg/ml) (μg/ml) 1 9.7 4.9 0 28.7 3.8 0 3 8.7 3.7 0 4 9.3 3.5 0 5 7.9 4.4 0 6 11.6 3.8 0 7 7 3.4 0 8 74.6 0 9 6.4 2.7 0 10 9.3 3.5 0 11 7.1 3 0 12 7.9 2.9 0 13 6.1 4 0 14 6.72.7 0 15 9 3.6 0 16 8.3 2.4 0 17 7.8 3.8 0 18 7.3 3 0 19 8.8 2.6 0 2010.2 4 0 21 9.4 3.6 0 22 7.4 3 0 23 7.5 3.6 0 24 8.6 3.3 0 25 7.8 3.3 026 8.9 3 0 27 8.1 3.3 0 28 11.1 3.3 0 29 8 3.9 0 30 5.7 4 0 31 9.1 3.5 032 8.1 4 0 33 9 4.3 0 34 8.1 3.3 5.4 35 8.5 2.9 0 36 7.7 3.4 0 37 8.32.6 0 38 8.3 3.6 0 39 11.2 3.2 0 40 9.1 3.1 41 10.7 3.5 0 42 9.6 3.1 043 8 3.3 0 44 15.6 3.5 0 45 10.2 3.6 0 46 8.1 3.9 0 47 8.4 2.5 0 48 11.83.1 0 49 7.5 3 0 50 9.9 3 0 51 8.1 3.4 0 52 9.7 3.2 0 53 7.8 3.4 0 548.7 3.4 0 55 7.9 3.5 0 56 8.4 3 0 57 9.4 3.5 0 58 9.5 3.2 0 59 9.3 3.2 060 7.8 3.4 0 61 7.9 3.2 0 62 8.9 3.8 0 63 8 3.1 0 64 7.7 3.3 0 65 10.13.5 0 66 5 5.2 17.1 67 5.5 4.5 0 68 6.2 4 0 69 6.6 3.8 0 70 8.4 3.6 0 719.9 4 0 72 6.7 4.4 0 73 6.1 3.4 0 74 6.5 3.2 0 75 8.8 2.9 0 76 5.2 3.7 077 7.1 4.2 0 78 5.6 3.2 0 79 11.3 7.6 1225 80 5.7 3.5 0 81 7.1 3.3 0 8210.2 3.6 0 83 8.8 3.8 0 84 6.7 3.5 0 85 5.2 4 0 86 10.4 3 0 87 5.3 3.48.6 88 7.9 4 0 89 9.9 3.6 0 90 8.2 3.8 0 91 8.9 4 0 92 6.4 3.8 0 93 8.26.6 1868 94 9.9 3.3 0 95 5.9 5.7 969 96 10.1 6.4 1010 97 6.8 4.1 0 986.3 4.5 0 99 6.5 4.7 0 100 10.3 7.5 1555 101 8.5 3.2 0 102 9.1 4.2 0 10310 3.9 0 104 6.7 3.7 0 105 9.4 5.5 21.1

REFERENCES

Used in Example 8:

-   1. Leucocyte counts in the healthy English Thoroughbred in Training.    Allen B V, Kane C E, Powell D G. Equine Veterinary Journal 1984;    16:207-209.-   2. Serum amyloid A protein (SAA) in horses: objective measurement of    the acute phase response.-   Pepys M B, Baltz M L, Tennent G A, Kent J, Ousey J, Rossdale P D.-   Equine Vet J. 1989 March; 21(2):106-9.-   3. Plasma fibrinogen as a parameter of the presence and severity of    inflammation in horses and cattle.-   van Wuijckhuise-Sjouke L A. Tijdschr Diergeneeskd. 1984 Nov. 1;    109(21):869-72.-   4. Acute phase response in the horse: plasma protein changes    associated with adjuvant induced inflammation.-   Patterson S D, Auer D, Bell K.-   Biochem Int. 1988 August; 17(2):257-64

The invention claimed is:
 1. A test fluid collection system for acollection of metered quantity of a test fluid to be diluted foranalysis, the system comprising: a housing comprising: i) a collectiontip; ii) a collection port; iii) a dispensing inlet; and iv) adispensing nozzle; wherein the collection tip is at a first end of thehousing and is adapted to contact a sample of the test fluid; whereinthe collection port is proximal to and in fluid communication with thecollection tip and comprises two spaced members having opposinghydrophilic surfaces; wherein said surfaces define a volume between themwhich corresponds to the metered quantity of test fluid to be collected;wherein the distance between the spaced members enables the test fluidfrom the sample tip to be drawn into the volume by capillary action;wherein the collection tip and dispensing nozzle are at opposite ends ofsaid housing; wherein the dispensing inlet is proximal to the collectiontip, and in fluid communication via a dispensing channel to thedispensing nozzle which runs through said housing; wherein the housingis adapted to be fitted into the opening of a liquid containercontaining liquid for diluting the metered quantity of the test fluid,with said collection port and said dispensing inlet within saidcontainer, and with a seal fit between said opening and said housing;and wherein the dispensing channel decreases in diameter between 10-21%over the height of the dispensing nozzle and wherein the dispensingnozzle is flared at the dispensing nozzle exit to dispense drops of thediluted test fluid in a uniform manner from the container when thevolume of said container is reduced and the diluted test fluid in saidcontainer is forced into said dispensing channel.
 2. The system asclaimed in claim 1, wherein the housing has a circular cross-section ofcircumference between 15 and 20 mm at the widest point.
 3. The system asclaimed in claim 1, wherein the collection tip is positioned on acollection stem, which stem incorporates the dispensing inlet and thecollection port.
 4. The system as claimed in claim 1, wherein thecollection stem is greater than or equal to about 20, 25, 30, 40, 45, or50 mm length.
 5. The system as claimed in claim 1, wherein the surfacesare parallel or are angled away from each other by about 1°, 2°, 3°, 4°,or 5°.
 6. The system as claimed in claim 1, wherein the surfaces arecoated with a hydrophilic coating or an anti-coagulant.
 7. The system asclaimed in claim 1, wherein the members are bevelled or shaped toincrease surface tension in the volume configured to hold the fluid. 8.The system as claimed in claim 1, wherein the members are two closelyaligned walls which form an open channel.
 9. The system as claimed inclaim 1, wherein the housing includes a fitting portion which is adaptedto be a push fit into the opening of the container and retained byfriction or compression.
 10. The system as claimed in claim 1, whereinthe housing includes a fitting portion which is adapted to be a push fitinto the opening of the container and retained by friction orcompression, and wherein the fitting portion is a cylindrical waistwhich optionally includes a frusto conical portion which is tapered tofacilitate insertion of the fitting portion into the opening of thecontainer.
 11. The system as claimed in claim 1, wherein the collectiontip is profiled and dimensioned to allow attachment of a luer endneedle, to wick a test fluid sample directly from the sample, or both.12. The system as claimed in claim 1, further comprising a liquidcontainer containing liquid for diluting the metered quantity of thetest fluid.
 13. The system as claimed in claim 1, wherein the dispensingchannel within the dispensing nozzle decreases in diameter by between20-21% over the height of the dispensing nozzle.